Micro-devices for disease detection

ABSTRACT

Among others, the present invention provides piezo-electric micro-devices for detecting at the microscopic level an electric, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, physical, bio-chemical, bio-physical, physical-chemical, bio-physical-chemical, bio-mechanical, bio-electro-mechanical, electro-mechanical, or mechanical property of the biologic subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/883,215, filed on Aug. 5, 2013, which is a national phaseapplication of PCT/U.S. 2011/054979, filed on Oct 5, 2011, which in turnclaims priority to U.S. Application No. 61/389,960, filed on Oct. 5,2010; U.S. Application No. 61/430,641, filed on Jan. 7, 2011; U.S.Application No. 61/467,097, filed on Mar. 24, 2011; U.S. Application No.61/498,954, filed on Jun. 20, 2011; and International Application No.PCT/U.S. 2011/042637, filed on Jun. 30, 2011, the contents of all ofwhich are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Many serious diseases with high morbidity and mortality, includingcancer and heart diseases, are very difficult to diagnose early andaccurately. Current disease diagnosis technologies typically rely onmacroscopic data and information such as body temperature, bloodpressure, and scanned images of the body. To detect serious diseasessuch as cancer, many of the diagnosis apparatus commonly used today arebased on imaging technologies, including x-ray, CT scan, and nuclearmagnetic resonance (NMR). While they provide various degrees ofusefulness in disease diagnosis, most of them cannot provide accurate,totally safe, and cost-effective diagnosis of such serious diseases ascancer at an early stage. Further, many of the existing diagnosistechniques and related apparatus are invasive and sometimes not readilyaccessible, especially in remote regions or rural areas.

Even the newly emerged DNA tests have not been proven effective indiagnosing a wide range of diseases in a rapid, reliable, accurate, andcost-effective manner. In recent years, there have been some efforts inusing nano technologies for various biological applications, with mostof the work focused on gene mapping and moderate developments in thefield of disease detection. For instance, Pantel et al. discussed theuse of a MicroEelectroMechanical Systems (MEMS) sensor for detectingcancer cells in blood and bone marrow in vitro (see, e.g., Klaus Pantelet al., Nature Reviews, 2008, 8, 329); Kubena et al. disclose in U.S.Pat. No. 6,922,118 the deployment of MEMS for detecting biologicalagents; and Weissman et al. disclose in U.S. Pat. No. 6,330,885utilizing MEMS sensor for detecting accretion of biological matter.

However, to date, most of the above described technologies have beenlimited to isolated examples for sensing, using systems of relativelysimple constructions and large dimensions but often with limitedfunctions, and lack sensitivities and specificities. Further, someexisting technologies utilizing nano-particles and biological approacheshave the drawbacks of requiring complicated sample preparationprocedures (such as using chemical or biological markers), difficulty indata interpretation, and too much reliance on visual and color change asmeans of diagnosis (which is subjective and of limited resolution),making them unsuitable for early stage disease detection, e.g., for suchserious diseases as cancer, and particularly for routine hospitalscreening and/or regular physical check-up examinations.

These drawbacks call for novel solutions that not only overcome them butalso bring enhanced accuracy, specificity, efficiency, non-invasiveness,practicality, simplicity, and speed in early-stage disease detection atreduced costs.

SUMMARY OF THE INVENTION

The present invention in general relates to a class of innovativedisease detection apparatus which utilizes novel micro-devices (orfunctionalities) integrated onto them for carrying out diagnosis atmicroscopic levels, in vivo or in vitro, on a single cell, a singlebiological molecular (e.g., DNA, RNA, or protein), a single biologicalsubject (e.g., a single virus), or other sufficiently small unit orfundamental biological composition. This class of apparatus can be madeby using state-of-the-art micro-device fabrication technologies andnovel process flows such as integrated circuit fabrication technologies.As used herein, the term “disease detection apparatus” can beinterchanged with such terms as disease detection device or apparatusintegrated with micro-devices, or any other similar terms of the samemeaning. Apparatus of this invention containing multiple micro-devicescan detect multiple parameters of a biological sample to be analyzed.These disease detection apparatus are capable of detecting diseases attheir early stages with a high degree of sensitivity, specificity,speed, simplicity, practicality, convenience (e.g., reduced apparatussize), or affordability (e.g., reduced costs).

One key component of the detection apparatus is a class of novelmicro-devices and their inventive fabrication processes which enablethese novel micro-devices to perform at a much higher level than thoseof conventional disease detection apparatus or technologies, due to muchimproved detection sensitivity, specificity, simplicity, practicality,and speed. Examples of fabrication techniques that can be used to makethe micro-devices described herein include but not limited tomechanical, chemical, physical-chemical, chemical mechanical,bio-physical, bio-physical mechanical, electro-mechanical,bio-electro-mechanical, micro-electro-mechanical,electro-chemical-mechanical, electro-bio-chemical-mechanical,nano-fabrication techniques, integrated circuit and semiconductormanufacturing techniques and processes. For a general description ofsome of the applicable fabrication technologies, see, e.g., R. Zaouk etal., Introduction to Microfabrication Techniques, in MicrofluidicTechniques (S. Minteer, ed.), 2006, Humana Press; MicrosystemEngineering of Lab-on-a-chip Devices, 1st Ed. (Geschke, Klank &Telleman, eds.), John Wiley & Sons, 2004. Micro-device functionalitieswould at least include sensing, detecting, measuring, diagnosing,monitoring, and analyzing for disease diagnosis. Multiple micro-devicescan be integrated onto a piece of detection apparatus to make theapparatus more advanced and sophisticated for further enhancedmeasurement sensitivity, specificity, speed and functionalities, withability to measure the same parameter or a set of different parameters.

Optional components of the apparatus includes means to perform at leastthe function of addressing, controlling, forcing, receiving, amplifying,manipulating, or storing information from each probe. Such means can be,e.g., a central control unit that includes a controlling circuitry, anaddressing unit, an amplifier circuitry, a logic processing circuitry, amemory unit, an application specific chip, a signal transmitter, asignal receiver, or a sensor.

Specifically, one aspect of this invention provides apparatus fordetecting a disease, each comprising a first micro-device and a firstsubstrate supporting the first micro-device, wherein the firstmicro-device contacts a biological subject to be analyzed and is capableof measuring at the microscopic level an electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,electro-mechanical, electro-chemical, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-electro-mechanical,bio-electro-chemical, bio-electro-chemical-mechanical, physical, ormechanical property of the biologic material. The apparatus can furtheroptionally include a device for reading the data from measuring theproperty.

In some embodiments, the difference in the measured property of thetested biologic material and that of a biologic sample from a subjectfree of the disease is indicative of the possible occurrence of thedisease in early stage.

In some other embodiments, the electrical property is surface charge,surface potential, oscillation in electrical signal (e.g., oscillationin ions, pulsing electrical field, pulsing surface charge, pulsingvoltage), electrical field, electrical field distribution, electricalcharge distribution, or impedance; the thermal property is temperature;the chemical property is pH value, ionic strength, bonding strength; thephysical property is density; and the mechanical property is hardness,shear strength, elongation strength, fracture stress, adhesion,elasticity, or density.

In some embodiments, the probing and detecting device applies to thebiological subject a voltage ranging from about 1 mV to about 10 V, orfrom about 1 mV to about 1.0 V.

In some embodiments, the first micro-device comprises a conductivematerial, an electrically insulating material, a biological material, ora semiconductor material.

In some other embodiments, each of the apparatus further comprises atleast one or more additional micro-devices. In these embodiments, eachof the micro-devices contained in the apparatus comprises a conductivematerial, an electrically insulating material, or a semiconductormaterial; and each of the micro-devices can comprise the same ordifferent material(s) and can measure the same or different propertiesat the same or different time.

In some embodiments, the probing device and the micro-devices are placedwith a desired distance between each other. These multiple micro-devicescan be spaced out, e.g., with a distance of at least 10 angstroms on thesubstrate, or with a distance ranging from about 5 microns to about 100microns.

The multiple micro-devices integrated in a disease detection apparatuscan sequentially and/or simultaneously measure various parameters from abiological subject being detected at macroscopic and/or microscopiclevels. Sometimes, in an apparatus with multiple micro-devices, somemicro-devices can act as probing devices to disturb the biologicalsubject and trigger a response from the biological subject, while othermicro-devices in the apparatus can act as detection devices to measurethe triggered response by the biological subject.

In some other embodiments, each of the micro-devices has the sizeranging from about 1 angstrom (Å) to about 5 millimeter (e.g., from 5 Åto 1 millimeter).

In some other embodiments, the apparatus comprises one or moreadditional substrates on which the micro-devices are placed. Each of thesubstrates can comprise the same or a different material (e.g., aconductive material or an insulator) and can be in the same or adifferent shape (e.g., a slab or a tube), and each substrate can be atwo- or three-dimensional object. They can take the form of cylinder,rectangle, cube, slabs, or any other desired shapes and configurations,in order to further improve their measurement sensitivity, specificity,speed, sample size, and reduce cost and size.

In terms of detection apparatus to integrate micro-devices, in one noveldetection apparatus design, to increase measurement sensitivity,micro-devices mounted on two slabs separated by a small spacing withsample to be measured between the two said slabs can be used to detectdisease with improved speed, with micro-devices measuring cells, DNAs,and desired items in the sample in parallel. The surface area of theslabs can be maximized in order to have maximum number of micro-devicesplaced on the slabs and enhance measurement efficiency and speed.Optionally, multiple micro-devices integrated on the surface of theslabs can be closely spaced with their spacing matching that of cells,DNAs, and items to be measured.

In another novel configuration, a detection apparatus integrated withmicro-devices is shaped in the form of a cylinder, with multiplemicro-devices with detection probes integrated/mounted in the intersurfaces of the cylinder and with sample to be measured (such as blood)flowing through the cylinder.

In yet another innovative configuration, a detection apparatus withintegrated micro-devices is shaped in the form of a rectangular pipe,with multiple micro-devices with detection probes integrated/mounted inthe inter surfaces of the pipe and with sample to be measured (such asblood) flowing through the rectangular pipe.

In another aspect, the invention provides another set of apparatus fordetecting a disease in a biological subject, comprising a system fordelivering the biological subject to be detected and a probing anddetecting device for probing and detecting the biological subject.

The difference in the measured property of the detected biologicmaterial and of a standard biologic sample is indicative of the possibleoccurrence of the disease.

In some embodiments, the probing and detecting device comprises a firstmicro-device and a first substrate supporting the first micro-device,the first micro-device contacts the biologic subject to be detected andis capable of measuring at the microscopic level an electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,electro-mechanical, electro-chemical, electro-chemical-mechanical,bio-chemical, bio-chemical-physical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property of thebiologic subject. For example, the electrical property can be surfacecharge, surface potential, resting potential, electrical current,electrical field distribution, electric dipole, electric quadruple,three-dimensional electrical or charge cloud distribution, electricalproperties at telomere of DNA and chromosome, or impedance; the thermalproperty can be temperature, or vibrational frequency of biological itemor molecules; the optical property can be optical absorption, opticaltransmission, optical reflection, optical-electrical property,brightness, or fluorescent emission; the chemical property can be pHvalue, chemical reaction, bio-chemical reaction, bio-electro-chemicalreaction, reaction speed, reaction energy, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, or bondingstrength; the physical property can be density or geometric size; theacoustic property is frequency, speed of acoustic waves, acousticfrequency and intensity spectrum distribution, acoustic intensity,acoustical absorption, or acoustical resonance; and the mechanicalproperty is internal pressure, hardness, shear strength, elongationstrength, fracture stress, adhesion, mechanical resonance frequency,elasticity, plasticity, or compressibility.

In some embodiments of the apparatus, the probing and detecting deviceapplies to the biological subject a voltage ranging from about 1 mV toabout 10 V, or from about 1 mV to about 1.0 V.

In some embodiments of the apparatus, the first micro-device comprises aconductive material, an electrically insulating material, a biologicalmaterial, or a semiconductor material.

In some embodiments of the apparatus, the first micro-device has a sizeranging from about 1 angstrom to about 5 millimeter.

In some embodiments of the apparatus, the probing and detecting devicefurther comprises one or more additional micro-devices, each of which isalso capable of measuring at the microscopic level an electric,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property of thebiologic entity. The electrical property can be surface charge, surfacepotential, resting potential, electrical current, electrical fielddistribution, electric dipole, electric quadruple, three-dimensionalelectrical or charge cloud distribution, electrical properties attelomere of DNA and chromosome, or impedance; the thermal property canbe temperature, or vibrational frequency of biological item ormolecules; the optical property can be optical absorption, opticaltransmission, optical reflection, optical-electrical property,brightness, or fluorescent emission; the chemical property can be pHvalue, chemical reaction, bio-chemical reaction, bio-electro-chemicalreaction, reaction speed, reaction energy, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, or bondingstrength; the physical property can be density or geometric size; theacoustic property can be frequency, speed of acoustic waves, acousticfrequency and intensity spectrum distribution, acoustic intensity,acoustical absorption, or acoustical resonance; and the mechanicalproperty can be internal pressure, hardness, shear strength, elongationstrength, fracture stress, adhesion, mechanical resonance frequency,elasticity, plasticity, or compressibility.

In some embodiments of the apparatus, each of the additionalmicro-devices comprises a conductive material, an electricallyinsulating material, a biological material, or a semiconductor material.Further, each of the additional micro-devices comprises a material thatis the same as or different from the material of the first micro-deviceand is capable of measuring the same or different property of thebiologic subject as the first-micro-device does.

In some embodiments of the apparatus, the first micro-device and each ofthe additional micro-devices are capable of measuring the surfacecharge, surface potential, resting potential, electrical current,electrical field distribution, electric dipole, electric quadruple,three-dimensional electrical or charge cloud distribution, electricalproperties at telomere of DNA and chromosome, impedance, temperature,vibrational frequency, optical absorption, optical transmission, opticalreflection, optical-electrical property, brightness, fluorescentemission, pH value, chemical reaction, bio-chemical reaction,bio-electro-chemical reaction, reaction speed, reaction energy, oxygenconcentration, oxygen consumption rate, ionic strength, catalyticbehavior, bonding strength, density, geometric size, frequency, speed ofacoustic waves, acoustic frequency and intensity spectrum distribution,acoustic intensity, acoustical absorption, acoustical resonance,internal pressure, hardness, shearing strength, elongation strength,fracture stress, adhesion, mechanical resonance frequency, elasticity,plasticity, or compressibility. They can measure the same or differentproperties at the same or different times.

In some embodiments of the apparatus, the probing device and themicro-devices are placed with a desired distance between each other.

In some embodiments of the apparatus, each of the additionalmicro-devices has a size ranging from about 1 angstrom to about 5millimeter.

In some embodiments of the apparatus, the micro-devices are spaced outon the substrate by a distance of at least 10 angstroms (e.g., fromabout 5 microns to about 100 microns).

In some embodiments of the apparatus, the substrate is in the shape of aslab, a rectangle, a cube, a tube, or an array of tubes; or thesubstrate is a three-dimensional object.

In some embodiments of the apparatus, the probing and detecting devicefurther comprises a second substrate of the same or different materialas the first substrate.

In some embodiments, the apparatus further comprises a device forreading the data from measuring the property by the probing anddetecting device.

In some embodiments, the apparatus each further comprises a system fordelivering a fluid, which comprises a pressure generator, a pressureregulator, a throttle valve, a pressure gauge, and distributing kits.The pressure generator can include a motor piston system and a bincontaining compressed gas; the pressure regulator can down-regulate orup-regulate the pressure to a desired value; the pressure gauge feedsback the measured value to the throttle valve, which then regulates thepressure to approach the target value.

The fluid to be delivered in the apparatus can be a liquid or gas.Examples of the liquid include blood, urine, saliva, tear, saline, andsweat; whereas examples of the gas include nitrogen, argon, helium,neon, krypton, xenon, or radon.

In some embodiments of the apparatus, the probing and detecting devicefurther comprises a system controller which comprises a pre-amplifier, alock-in amplifier, an electrical meter, a thermal meter, a switchingmatrix, a system bus, a nonvolatile storage device, a random accessmemory, a processor, or a user interface. The interface may include asensor which can be, e.g., a thermal sensor, a flow meter, an opticalsensor, or a sensor comprising one or more piezo-electric materials.

In some embodiments, the apparatus may further include a biologicalinterface, a system controller, or at least one system for reclaiming ortreatment medical waste. Reclaiming and treatment of medical waste isperformed by the same system or by two different systems.

In some embodiments, the apparatus further include a testing sampledelivery system, a testing sample distribution system, a distributionchannel, a pre-processing unit, a detection device, a global positioningsystem, a motion device, a signal transmitter, a signal receiver, asensor, a memory storage unit, a logic processing unit, an applicationspecific chip, a testing sample recycling and reclaiming unit, amicro-electro-mechanical device, a multi-functional device, or amicro-instrument to perform surgery, cleaning, or medical function. Suchadditional components each may be fabricated by methods known in theart, e.g., as described in PCT/U.S. 2010/041001, PCT/U.S. 2011/024672,U.S. application Ser. No. 12/416,280, and PCT/U.S. 2011/042637, all ofwhich are incorporated herein by reference in their entireties.

In some embodiments of the apparatus, the system for delivering thebiological subject comprises at least one channel inside which thebiological subject to be detected travels in a certain direction; theprobing and detecting device comprises at least one probing micro-deviceand at least one detecting micro-device, at least one probingmicro-device is located before at least one detecting micro-devicerelative to the direction in which the biological subject travels, andthe probing micro-device and the detecting micro-device can be attachedto the interior or exterior wall of the channel.

In some embodiments, the probing and detecting device comprise at leasttwo detecting micro-devices capable of measuring at the micro-level thesame or different properties of the biological subject.

In some further embodiments, the detecting micro-devices are capable ofmeasuring at the microscopic level the surface charge, surfacepotential, resting potential, action potential, electrical voltage,electrical current, electrical field distribution, electrical chargedistribution, electric dipole, electric quadruple, three-dimensionalelectrical or charge cloud distribution, electrical properties attelomere of DNA and chromosome, dynamic changes in electricalproperties, dynamic changes in potential, dynamic changes in surfacecharge, dynamic changes in current, dynamic changes in electrical field,dynamic changes in electrical voltage, dynamic changes in electricaldistribution, dynamic changes in electronic cloud distribution,impedance, temperature, vibrational frequency, optical absorption,optical transmission, optical reflection, optical-electrical property,brightness, fluorescent emission, pH value, chemical reaction,bio-chemical reaction, bio-electro-chemical reaction, reaction speed,reaction energy, speed of reaction, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, bonding strength,density, geometric size, frequency, speed of acoustic waves, acousticfrequency and intensity spectrum distribution, acoustic intensity,acoustical absorption, acoustical resonance, internal pressure,hardness, shear strength, elongation strength, fracture stress,adhesion, mechanical resonance frequency, elasticity, plasticity, orcompressibility.

In some embodiments of the apparatus, the shapes and sizes of differentsections of the channel can be the same or different; the width of thechannel ranges from about 1 nm to about 1 mm; the channel can bestraight, curved, or angled; the interior wall of the channel defines acircular, oval, or polygon space; the interior wall of the channeldefines a circular or rectangular space; the channel is a circularcarbon nano-tube.

In some embodiments of the apparatus, the carbon nano-tube has adiameter ranging from about 0.5 nm to about 100 nm and a length rangingfrom about 5.0 nm to about 10 mm.

In some embodiments of the apparatus, the interior wall of the channelhas at least one concave that may be in the same section as a probing ordetecting micro-device. The concave groove can be a cubic space or anangled space; the concave groove can have a depth ranging from about 10nm to about 1 mm.

In some embodiments of the apparatus, a distribution fluid is injectedinto the channel, either before or after the biological subject passes aprobing micro-device, to aid the traveling or separation of thebiological subject inside the channel. The distribution fluid can beinjected into the channel through a distribution fluid channel connectedto an opening in the channel wall.

In some yet other innovative embodiments, a cleaning fluid can be usedto clean the apparatus, particularly narrow and small spaces in theapparatus where biological residues and deposits (such as dried bloodand protein when they are used in or as a sample) likely accumulate andblock such spaces. Desired properties of such a cleaning fluid include,e.g., low viscosity and ability to dissolve the biological residues anddeposits.

The apparatus can be for detecting the diseases of more than onebiological subjects and the channel comprises a device located thereinfor separating or dividing the biological subjects based on differentlevels of a same property of the biological subjects. The separating ordividing device can be, e.g., a slit, and separates or dividesbiological subjects based on their properties such as surface charges.

The apparatus can further include a filtering device for removingirrelevant objects from the biologic subject for detection.

The biological subject can be a DNA, telomere of DNA, RNA, chromosome,cell, cell substructure, protein, tissue, or virus.

In some embodiments, the apparatus may further includes a unit fordelivering the biological subject, a channel, a detection unit, a datastorage unit, a data analysis unit, a central control unit, a biologicalsample recirculation unit, a waste disposal unit; a pre-processing unit,a signal processing unit, or a disposal processing unit. All theadditional components can be integrated on a single device or a boardalong with the delivering system and probing and detecting probe. Thepre-processing unit may comprise a sample filtration unit; a deliveryunit for delivering a desired ion, a biological component, or abio-chemical component; a recharging unit; a constant pressure deliveryunit; and a sample pre-probing disturbing unit. The sample filtrationunit may comprise an entrance channel, a disturbing fluid channel, anaccelerating chamber, and a slit. The signal processing unit maycomprise an amplifier, a lock-in amplifier, an A/D (analog-to-digital oralternative to direct electric current) converter, a micro-computer, amanipulator, a display, and network connections. The signal processingunit may collect more than one signal, collect multiple signalssimultaneously, collect signals simultaneously at different locations,and the signals can be integrated to cancel noise or to enhance thesignal to noise ratio. The collected signal(s) may also be processedthrough one or more lock-in amplifiers to enhance the signal to noiseratio, thereby improving detection sensitivity and repeatability.

In some embodiments of the apparatus, a bio-compatible fluid is injectedinto the disturbing fluid channel to separate the biological subject, orthe bio-compatible fluid is injected from the entrance of the disturbingfluid channel and delivered to an opening in the entrance channel wall.The biocompatible fluid comprises saline, water, an oxygen-rich liquid,or plasma.

In some embodiments of the apparatus, the angle between the entrancechannel and the disturbing fluid channel ranges from about 0° to about180°, from about 30° to about 150°, from about 60° to about 120°, orfrom about 75° to about 105°, or about 90°; the width of each channelranges from about 1 nm to about 1 mm; and at least one of the channelscomprises one probing device attached to the channel's sidewall, whereinthe probing device is capable of measuring at the microscopic level anelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject. The sample filtration unit may comprise an entrancechannel, a biocompatible micro-filter, or an exit channel.

In some embodiments of the apparatus, the biocompatible micro-filter iscapable of filtering the biological subject based on at least oneproperty selected from physical size, hardness, elasticity, shearstrength, weight, surface feature, optical, acoustical, thermal,chemical, mechanical, biological, bio-chemical, electrical,electro-chemical, magnetic, electromagnetic, electro-mechanical,electro-chemical-mechanical, and electro-chemical-biological property.

In some embodiments, at least one of the channels comprises at least twoprobing devices attached to the channel's sidewalls, and the probingdevices are capable of measuring at the microscopic level an electric,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, physical-chemical,bio-physical, bio-physical mechanical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject.

In some embodiments of the apparatus, the recharging unit rechargesnutrient or respiring gas to the biological subject. The nutrient caninclude a biocompatible strong or weak electrolyte, amino acid, mineral,ions, oxygen, oxygen-rich liquid, intravenous drip, glucose, or protein;and the respiring gas can include oxygen.

In some embodiments, the biological subject to be tested comprisesblood, urine, saliva, tear, saline, or sweat.

In some embodiments, the signal processing unit comprises an amplifier,a lock-in amplifier, an A/D converter, a micro-computer, a manipulator,a display, or a network connection. It can collect more than one signal,and the signals can be integrated to reduce (i.e., cancel out) noise andhence enhance the signal to noise ratio.

In still another aspect, the invention provides alternative apparatusfor detecting a disease in a biological subject. The apparatus eachcomprise a launching chamber to launch a probe object at a desired speedand direction, a detection unit, a probe object, a detection component,a channel for transporting the biological subject to be tested and theprobe object.

In some embodiments of these apparatus, the launching chamber comprisesa piston for releasing the probe object and a channel for directing theprobe object.

In some embodiments, the detection unit or the detection component iscapable of measuring at the microscopic level an electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,electro-mechanical, electro-chemical, electro-chemical-mechanical,bio-chemical, physical-chemical, bio-physical, bio-physical mechanical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject.

Yet still another set of apparatus for detecting a disease in abiological subject as provided by this invention are those fabricated bya method comprising: providing a substrate; sequentially depositing afirst material and a second material as two layers onto the substrate toform a material stack; patterning the second material to form a firstdesired feature; depositing a third material onto the material stack tocover the second material; optionally patterning the first material andthird material to form a second desired feature; and optionallydepositing a fourth material onto the material stack; wherein thedetection device is capable of interacting with the biological subjectto generate a response signal.

In some embodiments, in these methods used for fabricating theapparatus, the second material can be patterned by microelectronicprocesses.

In some embodiments, in these methods used for fabricating theapparatus, the first material and third material can be the same ordifferent.

In some embodiments, in these methods used for fabricating theapparatus, the first material and third material are patterned bylithography and etch processes selective to the second material to format least one type of trench feature in the layers of the third materialand first material.

In some embodiments, in these methods used for fabricating theapparatus, the fabrication method may further comprise capping the topof the material stack to form an enclosed trench. The enclosed trenchcan, e.g., be used to observe and record features and behaviors of thebiological subject. The capping can comprise, e.g., placing a seconddevice on the top of the material stack, and the second device can be adevice identical to the detection device being capped, a piece of glassor crystal, or a functional device selected from the group consisting ofan imaging device, an optical sensor, a memory storage, a signaltransmission, a logic processing component, a circuit for data storage,signal transmission, signal receiving, and signal processing.

In some embodiments, in these methods used for fabricating theapparatus, the first feature or second feature is selected from thegroup consisting of partitioned chambers, chambers connected withchannels, channels, probe generator (probe), detection probes,electrically connective interconnection lines, optical transmissionlines, and piezo-electric lines. For example, the partitioned chamberscan be for pre-processing of the biological subject for initialscreening and enhancement of concentration of diseased biologicalsubject for further testing, chambers connected with channels are forpre-processing and detection, channels can be for biological subject toflow through, the probe generator (probe) can be for generating probeand disturb signal onto the biological subject for triggering a responsesignal, the detection probe can be for measuring properties of thebiological subject and the response signal, the electrically connectiveinterconnection lines can be for transmitting signals, the opticaltransmission lines can be for transmitting signals, and piezo-electriclines can be for using piezo-electric effect to probe biologicalsubjects.

In some embodiments, in these methods used for fabricating theapparatus, the second material is patterned using lithography and etchprocesses selective to the first material to form a desired componentsuch as a detection probe.

In some embodiments, in these methods used for fabricating theapparatus, the first and third materials are patterned using lithographyand etch processes selective to the second material to form at least onetype of trench feature in the layers of the third and first materials,with the second material reasonably aligned with the wall of the trench.

In some embodiments, in these methods used for fabricating theapparatus, the thickness of the fourth material is thinner than that ofthe third material.

In some embodiments, the second and the fourth materials form detectionprobes.

In some embodiments, the second and the fourth materials form a probeand a detector, respectively.

In some embodiments, the apparatus may further include an imaging devicefor observing and recording properties and behaviors of the biologicalsubject.

In some embodiments, the apparatus may further include a pre-processingunit with chambers for pre-screening and enhancing a diseased biologicalsubject for further testing, channels for carrying fluidic sample toflow through, probes for probing and disturbing the biological subjectbeing tested for generating response signals, detection probes formeasuring properties and response signals of the biological subject, andan imaging device, a camera, a viewing station, an acoustic detector, athermal detector, an ion emission detector, or a thermal recorder forobserving and recording properties and behaviors of the biologicalsubject.

In some embodiments, the apparatus may further include a memory storageunit, a signal transmission component, a logic processing component, ora circuit for data storage, signal transmission, signal receiving, orsignal processing. These additional devices can be fabricated bymicroelectronics processes on the substrate where the first material isdeposited.

In some embodiments, the apparatus may have typical channel dimensionsranging from about 2 microns×2 microns to about 100 microns×100 micronsin cross sectional area for a square-shaped channel, a rectangle-shapedchannel, a radius ranging from about 1 micron to about 20 microns incross sectional area for a circular shaped channel, and a typical probedimension ranging from about 0.5 micron×0.5 micron to about 20microns×20 microns in cross sectional area for a square-shaped probe.

In some embodiments, the apparatus may have typical channel dimensionsranging from about 6 microns×6 microns to about 14 microns×14 microns incross sectional area for a square-shaped channel, a radius ranging fromabout 3 microns to about 8 microns in cross sectional area for acircular shaped channel, and a typical probe dimension ranging fromabout 0.5 micron×0.5 micron to about 10 microns×10 microns in crosssectional area for a square shaped probe.

In some embodiments, the first material and the fourth material eachcomprise un-doped oxide (SiO₂), silicon nitride, doped oxide, a polymermaterial, glass, or an insulating material.

In some embodiments, the second material and third material eachcomprise an electrically conductive material, aluminum, an aluminumalloy, copper, a copper alloy, tungsten, a tungsten alloy, gold, a goldalloy, silver, a silver alloy, or a piezo-electric material. Examples ofthe piezo-electric material include, but are not limited to, quartz,berlinite, gallium, orthophosphate, GaPO₄, tourmaline, ceramics, barium,titanate, BatiO₃, lead zirconate, titanate PZT, zinc oxide, aluminumnitride, and a polyvinylidene fluoride.

In yet some other embodiments, the second material and fourth materialeach comprise an electrically conductive material or a piezo-electricmaterial. Examples of the electrically conductive material include, butare not limited to, aluminum, an aluminum alloy, copper, a copper alloy,tungsten, a tungsten alloy, gold, a gold alloy, silver, a silver alloy;whereas examples of the piezo-electric material include, but are notlimited to, quartz, berlinite, gallium, orthophosphate, GaPO₄,tourmaline, ceramics, barium, titanate, BatiO₃, lead zirconate, titanatePZT, zinc oxide, aluminum nitride, and a polyvinylidene fluoride.

In some embodiments of the apparatus, the detection device comprises atleast one probe, at least one detector, or at least one pair of probeand detector, the probe generates a probing or disturbing signal ontothe biological subject to give a response signal, and the detectormeasures the response signal thus generated.

In some embodiments of the apparatus, the second material is patternedby microelectronic processes to form a first desired feature; the firstmaterial and third material are optionally patterned by microelectronicprocesses to form a second desired feature; and the first material andthird material can be the same or different.

In some embodiments, the methods for fabricating the apparatus furtherinclude capping the top of the material stack to form an enclosedtrench, with such trench used for test sample transportation ordetection site.

One of the key novel aspects of this patent application is the designand fabrication process flows of micro-devices and methods of using themicro-devices for contacting and measuring properties, at microscopiclevels and in a three dimensional space, of a biological subject (e.g.,a single cell or a single biological molecule such as DNA or RNA). Themicro-devices have micro-probes arranged in a three dimensional mannerwith feature sizes as small as a cell, a DNA, and a RNA and capable oftrapping, sorting, probing, measuring, manipulating, or modifyingbiological subjects.

Another aspect of this invention relates to methods for fabricating amicro-device. The methods include depositing various materials on asubstrate and, in the interims of depositing every two materials,pattern the materials by microelectronic technology and associatedprocesses, wherein the micro-device is capable of measuring at themicroscopic level the electric, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, physical, physical-chemical,bio-chemical, bio-physical, mechanical, bio-chemical mechanical,bio-electro-mechanical, bio-electro-chemical mechanical,electro-chemical mechanical, micro-electro-mechanical property of abiologic subject that the micro-device is to contact.

Still another aspect of this invention relates to methods forfabricating a micro-device, which include depositing a first material onthe substrate, pattering the first material by a microelectronic processto give rise to at least one patterned residual and leaving part of thesubstrate surface uncovered by the first material, depositing a secondnon-conductive material atop the processed first material and thesubstrate, creating an opening in the second material and exposing partof the patterned residual of the first material, filling up the openingin the second material with a third material. In some embodiments, themicroelectronic process comprises thin film deposition,photolithography, etching, cleaning, or chemical mechanical polishing.

Yet in still another aspect, the invention provides methods forfabricating a micro-device, which include the first step of depositing afirst material onto a substrate; the second step of depositing a secondmaterial onto the first material and then patterning the second materialwith a microelectronic technology or process; and repeating the secondstep at least once with a material that can be the same as or differentfrom the first or second material. The materials used in the repeatedsteps can be the same as or different from the first or second material.In some embodiments, at least one of the materials used in fabricatingthe micro-device is a piezo-electric material or a conductive material.

In some embodiments, multiple fabricated micro-devices can be coupled,joined, connected, and integrated by physical or electrical method toconstitute the more advanced devices.

In some embodiments, the apparatuses of this invention can be integratedon a single device (e.g., by using a semiconductor processingtechnology) or assembled on a board (e.g., by using a computer packagingtechnology).

In some embodiments, patterning of a material is done by amicroelectronic process (e.g., chemical vapor deposition, physical vapordeposition, or atomic layer deposition to deposit various materials on asubstrate as an insulator or conductor; lithography or etch to transferpatterns from design to structure; chemical mechanical planarization,chemical cleaning for particle removal, thermal spiking anneal to reducethe crystal defects, diffusion or ion implantation for doping elementsinto specific layers). In some embodiments, patterning is planarizationby chemical polishing, mechanical polishing, or chemical mechanicalpolishing.

In some other embodiments, the methods further include removal of astack of multiple layers of materials by wet etch, plasma etch, or byvapor etch.

In some embodiments, the micro-device can move in any direction. Forinstance, two micro-devices can move in opposite directions.

In some embodiments, the micro-device thus fabricated is so patternedthat it is capable of trapping, sorting, probing, measuring, ormodifying a biological subject; or that it can piece through themembrane of a cell.

Still another aspect of the invention relates to methods for fabricatinga device or apparatus for detecting disease in a biological subject,which include providing a substrate, sequentially depositing a firstmaterial and a second material as two different layers onto thesubstrate to form a material stack, patterning the second material bymicroelectronic processes to form a first desired feature, depositing athird material onto the material stack, optionally patterning the firstand third materials by microelectronic processes to form a seconddesired feature, and optionally depositing a fourth material onto thematerial stack.

In some embodiments, the methods further include steps of fabricating(utilizing processes including but not limited to depositing,patterning, polishing, and cleaning) additional components onto thesubstrate before sequentially depositing the first material and thesecond material as layers onto the substrate, wherein the additionalcomponents comprise a data storage component, a signal processingcomponent, a memory storage component, a signal transmitting component,a logic processing component, or an RF (radio-frequency) component.

In some other embodiments, the methods further include steps offabricating at least a circuit onto the substrate before sequentiallydepositing the first material and the second material as layers onto thesubstrate, wherein the circuit comprises a data storage circuit, asignal processing circuit, a memory storage circuit, a signaltransmitting circuit, or a logic processing circuit.

In still some other embodiments, the methods of this invention furtherinclude a step of planarizing the third material using chemicalmechanical polishing process or an etch back process, after the step ofdepositing the third material onto the material stack and before thestep of patterning the first and the third materials.

Examples of the suitable microelectronic processes include, but are notlimited to, thin film deposition, lithography, etch, polishing,cleaning, ion implantation, diffusion, and packaging as typically usedin microelectronics.

The first and third materials can be the same or different. They can be,for example, electrically insulating material, such as oxide, dopedoxide, silicon nitride, or a polymer.

The second material can be an electrically conductive material, apiezo-electric material, a semiconductor material, a thermal sensitivematerial, an optical material, a pressure sensitive material, an ionemission sensitive material, or any combination thereof. For example,the second material can be copper, aluminum, tungsten, gold, silver,glass, an aluminum alloy, a copper alloy, a tungsten alloy, a goldalloy, a silver alloy, quartz, berlinite, gallium, orthophosphate,GaPO₄, tourmaline, ceramics, barium, titanate, BatiO₃, lead zirconate,titanate PZT, zinc oxide, aluminum nitride, and a polyvinylidenefluoride.

In some embodiments, the first desired feature can be a probe, whereasthe second desired feature can be a recessed form, or a trench form inthe layers of the first and third materials.

In yet some other embodiment, the methods of this invention furthercomprise depositing a fourth material onto the material stack and thenpatterning the fourth material to form a recessed area such as a hole ata selected location.

In still another embodiment, the methods of this invention furthercomprise a step of removing the third material from the material stackby wet or vapor etch to form a detection chamber between the fourthmaterial and the substrate. Furthermore, they may also include a step ofremoving the first material from the material stack by wet or vapor etchto form a channel. The channel can connect the formed detection chamberwith additional chambers.

In yet still another embodiment, the methods of this invention furtherinclude a step of sealing or capping the top of the material stack toform an enclosed trench. In one example of this step, the top of thematerial stack is sealed or capped with an additional device onto thematerial stack. Examples of such an additional device include, but arenot limited to, an imaging device, a communication device, and adetecting probe. The above said device on top of the material stackcomprises of optical device, imaging device, camera, viewing station,acoustic detector, thermal detector, ion emission detector, and thermalrecorder.

In yet another aspect, the present invention provides methods forfabricating a device for detecting disease in a biological subject,which include providing a substrate, sequentially depositing a first anda second materials as layers onto the substrate to form a materialstack, patterning the second material by lithography and etch process toform a recessed area in the layer of the second material, depositing athird material onto the material stack, removing a portion of the thirdmaterial above the second material by etch back or polishing process,patterning the third material by lithography and etch to form at least aportion of recessed area in the layer of the third material, depositinga fourth material onto the material stack, and removing the portion ofthe fourth material above the third material by etch back or polishingprocess to keep at least a portion of the second and fourth material inthe same layer.

If desired, more layers of different materials can be deposited,patterned, cleaned, or planarized to form additional structures withmore features, functionalities, and complexities.

The first and third materials used in the methods of this invention canbe the same or different. In some embodiments, they are the same. Theycan be, e.g., an electrically insulating material. Examples of the firstand third materials include, but are not limited to, oxide, doped oxide,silicon nitride, or a polymer.

In some embodiments, following the deposition and processing of thethird or fourth material, at least one more material is deposited andprocessed to form a top layer with a detection chamber or channelsformed underneath.

Examples of the second material include, but are not limited to,electrically conductive materials, piezo-electric materials,semiconductor materials, thermal sensitive materials, a pressuresensitive material, an ion emission sensitive material, opticalmaterials, or any combinations thereof.

In some embodiments, a novel detection apparatus comprising a detectionchamber and/or channels for test sample transport is formed by methodsthat include the steps of: depositing a first material, patterning thefirst material (“material A”) to form at least a recessed area,depositing a second material (“material B”), removing the secondmaterial (“material B”) from areas above the first material (“materialA”) by using polishing and/or etch back processes, leaving the secondmaterial (“material B”) in the recessed area in the first materiallayer, depositing a third material (“material C”) to cover the firstmaterial (“material A”) and the second material (“material B”),patterning the third material (“material C”) to form at least a holesmaller than the recessed area(s) in the third material layer and aboveit, removing the second material (“material B”) optionally by usingvapor etch or wet etch, forming an enclosed cavity in the first materiallayer.

In addition to novel micro-devices and manufacturing process forfabricating them, packaging of such devices are also critical (a) inensuring its proper function and (b) how to incorporate (transport itinto the micro-device) biological sample into the micro-device fordetection, probing, communicating, and possibly manipulating, modifyingand treating such biological subjects. Specifically, after beingfabricated, the micro-devices typically need to be packaged forprotection from outside environment and for configuration for connectionwith the outside world (e.g., by electrical connection).

In this application, a set of novel designs, configurations, processes,and materials are disclosed, with the goals of protecting themicro-devices, connecting to the outside world, and transportingbiological samples into the micro-devices properly and effectively. Insome embodiments relating to this aspect, after being fabricated, amicro-device can be wrapped with a packaging material that forms aprotective or packaging layer around the micro-device. The packagingprocess may also allow for forming lead pins on the packaging materialfor connections (e.g., magnetic or electric connection) with outsidedevices, e.g., for data transmissions and instruction communications.The packaging material can be an organic polymeric material, aninorganic polymeric material, or a molding compound.

In some other embodiments, a novel cavity can be formed in the packagingor protective layer, which has at least one opening connecting to theinlet of the micro-device and at least one other opening connecting toan outside device such as an injection device. In this way, a biologicalsample can be injected into the cavity through the opening (e.g., byconnecting to an injector) and transported into the micro-device throughthe other opening connecting to the micro-device inlet.

In still some other applications, an outside device such as an injectiondevice can be directly connected to an inlet of, or fitted into, themicro-device for transporting a biological sample. In this case, it isimportant that the inlet is leak free at both ends connected to themicro-device and to the outside device (such as an injector). To achievethis, a first material with substantially high viscosity can be usedfirst to seal seams and cracks between the inlet and the micro-device,or between the outside device and the micro-device. It could be a solidmaterial or a material with very high viscosity. To secure its stabilityand resolve possible adhesion issues with the first material and thedevice, a second material (e.g., a material that has a lower viscosityand is sticky in nature, when melt or solution) can be applied. Examplesof such a material include epoxies, adhesives, and glues. To speed upthe drying process of the second material when it is in a solution, heatcan be applied (for example, an air flow at a temperature of 40° C. orhigher).

In yet some other embodiments, a novel detection apparatus can beintegrated with at least one micro-injector and at least one detector,in which the micro-injector can inject a desired object into thebiological subject to be tested to generate a response by the biologicalsubject and the detector detects the response thus generated by thebiological subject.

The invention further provides methods for detecting a biologicalsubject's dynamic response to a signal. These methods include providingan apparatus comprising two micro-devices of which one is a probingmicro-device and the other is a detecting micro-device and positionedwith a distance from the probing micro-device; contacting the biologicalsubject with the probing micro-device whereby the probing micro-devicemeasures a property of the biological subject at the microscopic levelor sends a stimulating signal to the biological subject; and thedetecting micro-device measures the response of the biological subjectthrough measuring properties of the biological subject at themicroscopic level. Optionally, the detecting micro-device contacts thebiological subject during the measurements.

In some embodiments, the signal is an electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,electro-mechanical, electro-chemical, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-electro-mechanical,bio-electro-chemical, bio-electro-chemical-mechanical, physical, ormechanical signal.

In some other embodiments, the property at the microscopic level is anelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-chemical-physical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property.

Examples of the electrical properties include, but are not limited to,surface charge, surface potential, resting potential, electricalcurrent, electrical field distribution, electric dipole, electricquadruple, three-dimensional electrical and/or charge clouddistribution, electrical properties at telomere of DNA and chromosome(also called sticky end or DNA end) or impedance. Examples of thethermal properties include temperature, and vibrational frequency ofbiological item and molecules. Examples of the optical propertiesinclude optical absorption, optical transmission, optical reflection,optical-electrical properties, brightness, and fluorescent emission.Examples of the chemical properties include pH value, chemical reaction,bio-chemical reaction, bio-electro-chemical reaction, reaction speed,reaction energy, speed of reaction, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, and bondingstrength. Examples of the physical properties include density andgeometric size. Examples of the acoustic properties include frequency,speed of acoustic waves, acoustic frequency and intensity spectrumdistribution, acoustic intensity, acoustical absorption, and acousticalresonance. Examples of the mechanical property include internalpressure, hardness, shear strength, elongation strength, fracturestress, adhesion, mechanical resonance frequency, elasticity,plasticity, and compressibility. The date from measuring one or more ofthe properties at the microscopic level can be used for detectingdiseases, e.g., cancer at its early stage, or for estimating the lifeexpectancy of the carrier of the biological subject.

In some other embodiments, the apparatus further includes a thirdmicro-device that is different from the probing micro-device and thedetecting micro-device; and the third micro-device measures the same ora different property of the biological subject as the probingmicro-device and the detecting micro-device do.

In still some other embodiments, the apparatus further includes a clockmicro-device that is different from the probing micro-device and thedetecting micro-device; and the type of clock micro-device is placed ata fixed distance before the probing micro-devices and detectingmicro-devices with a distinctive signal when a biological subject passesit and acts as a clock device.

Yet still in some embodiments, the data recorded by the detectingmicro-device is filtered by a phase lock-in technology to remove noiseunsynchronized to the clock signal in order to enhance signal to noiseratio and improve measurement sensitivity.

Another aspect of this invention relates to methods for detectingdisease in a biological subject, comprising providing an apparatuscomprising a channel, a detection probe, imaging device, a memorystorage component, a signal transmitting component, a signal receivingcomponent, or a logic processing component, pre-processing thebiological subject to enhance its concentration, measuring theproperties of the biological subject, optionally contacting thebiological subject with the probing component (probing micro-device orprobing tip) through the channel to trigger or result in a responsesignal, using the detection probe (e.g., detection micro-device ordetection component) to detect the response signal from the biologicalsubject, optionally separating diseased biological subject from healthybiological subject based on the response signal, optionally sending theseparated, suspected diseased biological subject on for further tests,and analyzing the response signal and reaching a diagnosis conclusion.The biological subject can be a DNA, a sub-structure in a cell, a cell,or a protein.

In some embodiments, the methods of this invention further includedetection of the response signal and behaviors of interaction or eventsoccurred between at least two biological subjects or at least onebiological subject with at least one non-biological subject. The atleast two biological subject can be different or identical, in type ofcomposition. Examples of interactions or events occurred between atleast two biological subjects include, but are not limited to, a DNAcolliding with another DNA, a cell smashing into another cell, a DNAcrashing into a cell, a protein colliding with another protein, or a DNAcrashing into a protein. Examples of interactions or events occurredbetween at least one biological subject with at least one non-biologicalsubject include, but not limited to, an inorganic particle collidingwith a biological subject, an organic particle colliding with abiological subject, or a composite particle colliding with a biologicalsubject.

Examples of the response signals include, but are not limited to, anelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-chemical-physical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, and mechanical signal.

Anther aspect of the current invention relates to methods for detectingdisease in a biological subject. The methods include providing anapparatus comprising a pre-processing unit, at least one detectiondevice, a partitioned chamber with channels connecting them, and aninjection device (for, e.g., injecting a probe material into thebiological subject to be tested), and measuring response signals fromthe biological subject, wherein the probe material comprises an organicparticle, an inorganic particle, a biological subject, or acomposite-based object.

Yet another aspect of the current invention relates to methods fordetecting a disease in a biological subject by interacting it with aprobe object, comprising providing an apparatus comprising a launchingchamber, a detection unit, and channels, launching a probe object ontothe biological subject, causing a collision between the probe object andthe biological subject to give rise to a response signal, recording anddetecting the response signal during and after the collision. The probeobject may comprise an organic particle, an inorganic particle, abiological subject, or a composite-based object.

Still another aspect of this invention relates to methods for detectinga disease in early stage in a biological subject. These methods includethe steps of collecting a first sample (including a cell or a biologicalmolecule) of the biological subject's tissue or organ potentiallycarrying the disease, collecting a second sample of the same tissue ororgan from a second subject free of the disease, separately contactingthe first and second samples with a disease detection apparatus of thisinvention, and comparing the data from the measurements of the first andsecond samples. As mentioned above, a disease detection apparatus ofthis invention includes a micro-device and a substrate supporting themicro-device, wherein the micro-device is capable of measuring at themicroscopic level the electric, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, physical, or mechanicalproperty of a biological sample.

Still a further aspect of this invention relates to a method of cellularcommunication. The micro-device can generate artificial microscopiccalcium (or other elements) oscillations which simulate theintracellular biological communications. This artificial signal can becoded to interact with cellular proteins, nucleus, and eventuallyregulates cell's determination or fate, which in turn can result incommunication, probing, modifying, manipulating, or control of abiological subject at the cellular level, hence giving rise to diagnoseor cure of diseases at the cellular level or in their early stage.

Yet still a further aspect of this invention relates to methods fordetermining cellular or molecular response to a signal. The methodsinclude the step of contacting a cell or biological molecule with adisease detection apparatus of this invention—which includes a firstmicro-device, a second micro-device, and a first substrate supportingthe first micro-device and second micro-device. The first micro-devicein the apparatus is capable of measuring at the microscopic level anelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property of thecell; and the second micro-device contacts the cell or biologicalmolecule and stimulates it with a signal.

In some embodiments of these methods, the apparatus further comprises athird micro-device that is capable of measuring at the microscopic levelthe same electric, magnetic, electromagnetic, thermal, optical,acoustical, biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property of thecell or biological molecule as the first micro-device is.

In some other embodiments, the cell contacts the first micro-device,second micro-device, and third micro-device in the order.

In some further embodiments, the signal is an electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,electro-mechanical, electro-chemical, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-electro-mechanical,bio-electro-chemical, bio-electro-chemical-mechanical, physical, ormechanical signal.

In some embodiments of the apparatus of this invention, the system fordelivering the biological subject includes at least one channel insidewhich the biological subject to be detected travels in a certaindirection; the probing and detecting device includes at least oneprobing micro-device and at least one detecting micro-device, at leastone probing micro-device is located before at least one detectingmicro-device relative to the direction in which the biological subjecttravels, and the probing micro-device and the detecting micro-device canbe attached to the interior or exterior wall of the channel. In someother embodiments, multiple channels with different geometries areutilized.

In some examples of these embodiments, the probing and detecting deviceincludes at least two detecting micro-devices capable of measuring atthe micro-level the same or different properties of the biologicalsubject. Examples of the electrical properties include, but are notlimited to, surface charge, surface potential, resting potential,electrical current, electrical field distribution, electric dipole,electric quadruple, three-dimensional electrical and/or charge clouddistribution, electrical properties at telomere of DNA and chromosome orimpedance; examples of the thermal properties include temperature, andvibrational frequency of biological item and molecules; examples of theoptical properties include optical absorption, optical transmission,optical reflection, optical-electrical properties, brightness, andfluorescent emission; examples of the chemical properties include pHvalue, chemical reaction, bio-chemical reaction, bio-electro-chemicalreaction, reaction speed, reaction energy, speed of reaction, oxygenconcentration, oxygen consumption rate, ionic strength, catalyticbehavior, and bonding strength; examples of the physical propertiesinclude density and geometric size; examples of the acoustic propertiesinclude frequency, speed of acoustic waves, acoustic frequency andintensity spectrum distribution, acoustic intensity, acousticalabsorption, and acoustical resonance; and examples of the mechanicalproperty include internal pressure, hardness, shear strength, elongationstrength, fracture stress, adhesion, mechanical resonance frequency,elasticity, plasticity, and compressibility. For instance, the detectingmicro-devices are capable of measuring at the microscopic level thesurface charge, electric potential, brightness, fluorescent emission,geometric size, shape, frequency, internal pressure, or temperature ofthe biological subject.

In some other embodiments, the shapes and sizes of different sections ofthe channel can be the same or different; the width of the channel canbe about 1 nm˜1 mm (e.g., 1˜750 nm, 1˜600 nm; 100˜800 nm, 200˜750 nm, or400˜650 nm); the channel can be straight, curved, or angled; theinterior wall of the channel defines a circular, oval, or polygon (e.g.,rectangular) space.

An example of a suitable channel is a circular carbon nano-tube, whichcan have a diameter of, e.g., about 0.5˜100 nm, or a length of, e.g.,about 5.0 nm˜10 mm.

In some embodiments, the interior wall of the channel has at least oneconcave that may be in the same section as a probing or detectingmicro-device. The concave groove can be, e.g., a cubic space or anangled space. It can have a depth of, e.g., about 10 nm˜1 mm.

In some other embodiments, a distribution fluid can be injected into thechannel, either before or after the biological subject passes a probingmicro-device, to aid the traveling or separation of the biologicalsubject inside the channel. A suitable distribution fluid is abiocompatible liquid or solution, e.g., water or saline. Thedistribution fluid can be injected into the channel through adistribution fluid channel connected to an opening in the channel wall.Utilizing such a distribution fluid allows, among others, preparation ofthe surface of the channel (in which the biological subject travels),cleaning of the channel, disinfection of the apparatus, and enhancingthe measurement sensitivity of the apparatus.

In yet some other embodiments, a cleaning fluid can be used to clean anapparatus of this invention, particularly narrow and small spaces in theapparatus wherein biological residues and deposits (e.g., dried blood orprotein when it is used as or contained in a sample to be tested by theapparatus) are likely to accumulate and block such spaces. Desiredproperties of such a cleaning fluid include low viscosity and ability todissolve the biological residues and deposits. For example, when anapparatus of this invention is used for detecting a disease, certainbiological samples, such as blood, could result in blockage to narrow,small spaces in the apparatus such as narrow channels when the blood isallowed to dry. The cleaning solution is expected to address this issueby dissolve the biological samples.

In still some other embodiments, the apparatus of this invention can befor detecting the diseases of more than one biological subject, and thechannel comprises a device located therein for separating or dividingthe biological subjects based on different levels of a same property ofthe biological subjects. An example of such a separating or dividingdevice is a slit that can, e.g., separate or divide biological subjectsbased on their surface charges, their density, their size, or otherproperties such as electrical, thermal, optical, chemical, physical,magnetic, electromagnetic, and mechanical properties. Examples of theelectrical properties include, but are not limited to, surface charge,surface potential, resting potential, electrical current, electricalfield distribution, electric dipole, electric quadruple,three-dimensional electrical and/or charge cloud distribution,electrical properties at telomere of DNA and chromosome or impedance;examples of the thermal properties include temperature, and vibrationalfrequency of biological item and molecules; examples of the opticalproperties include optical absorption, optical transmission, opticalreflection, optical-electrical properties, brightness, and fluorescentemission; examples of the chemical properties include pH value, chemicalreaction, bio-chemical reaction, bio-electro-chemical reaction, reactionspeed, reaction energy, speed of reaction, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, and bondingstrength; examples of the physical properties include density andgeometric size; examples of the acoustic properties include frequency,speed of acoustic waves, acoustic frequency and intensity spectrumdistribution, acoustic intensity, acoustical absorption, and acousticalresonance; and examples of the mechanical property include internalpressure, hardness, shear strength, elongation strength, fracturestress, adhesion, mechanical resonance frequency, elasticity,plasticity, and compressibility.

In yet still some other embodiments, the apparatus of this invention canfurther include a filtering device for removing irrelevant objects fromthe biologic subject for detection.

In another aspect, the invention provides methods for obtaining dynamicinformation of a biologic material, each comprising contacting thebiological subject (e.g., including but not limited to a cell,substructure of a cell such as cell membrane, a DNA, a RNA, a protein,or a virus) with an apparatus comprising a first micro-device, a secondmicro-device, and a first substrate supporting the first micro-deviceand second micro-device, wherein the first micro-device is capable ofmeasuring at the microscopic level an electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,physical, or mechanical property of the biological subject, and thesecond micro-device contacts the biological subjects and stimulates itwith a signal.

In yet another embodiment, the micro-device in the detection apparatuscan communicate with biological subjects such as cells, DNA, RNA, virus,or protein. Further, the micro-device can trap, sort, analyze, treat,and modify biological subjects such as cells, DNA, RNA, blood cells,protein, or virus. Specifically, an array of micro-devices arranged in adesired manner can trap, sort, detect, and modify DNA structures.

In some embodiments, the apparatus further comprising a thirdmicro-device that is capable of measuring at the microscopic level thesame electric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, bio-chemical, physical, or mechanical property ofthe cell as the first micro-device is. In some other embodiments, thecell contacts the first micro-device, second micro-device, and thirdmicro-device in the order. In still some other embodiments, the signalis an electric signal, a magnetic signal, an electromagnetic signal, athermal signal, an optical signal, an acoustical signal, a biologicalsignal, a chemical signal, a physical signal, or a mechanical electricsignal.

In another aspect, this invention provides alternative methods fordetecting a biological subject's dynamic information. The methods eachinclude providing an apparatus comprising a clock micro-device, aprobing micro-device, and a first detection micro-device, with theprobing micro-device being placed between the clock micro-device and thedetection micro-device; contacting the biological subject with the clockmicro-device whereby the clock micro-device registers the arrival of thebiological subject, and optionally measures a property of the biologicalsubject at the microscopic level; contacting the biological subject withthe probe device with a periodic probe signal delivered onto thebiological subject; using the detecting micro-device to detect responsesignal from the biological subject; and processing the detected signalby the detection micro-device using phase lock-in technology to filterout signal components un-synchronized to the frequency of the probesignal, and amplify the signal synchronized to the probe signal.

In some embodiments of these methods, there is a distance of at least 10angstroms between the clock micro-device and the first detectingmicro-device.

In some other embodiments, the response signal is an electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,electro-mechanical, electro-chemical, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-electro-mechanical,bio-electro-chemical, bio-electro-chemical-mechanical, physical, ormechanical signal.

In some other embodiments, the first probing micro-device optionallymeasures the same property of the biological subject at the microscopiclevel as the first detecting micro-device does.

In still some other embodiments, the apparatus used in the methodsfurther comprises a second probing micro-device capable of sending astimulating signal to the biological subject that is different from thesignal sent by the first probing micro-device.

In still some other embodiments, the apparatus used in the methodsfurther comprise a second detecting micro-device capable of measuringthe same property of the biological subject at the microscopic level asthe first detecting micro-device does.

In yet still some other embodiments, the electrical property is surfacecharge, surface potential, resting potential, electrical current,electrical field distribution, electric dipole, electric quadruple,three-dimensional electrical or charge cloud distribution, electricalproperties at telomere of DNA and chromosome, or impedance; the thermalproperty is temperature, or vibrational frequency of biological item ormolecules; the optical property is optical absorption, opticaltransmission, optical reflection, optical-electrical property,brightness, or fluorescent emission; the chemical property is pH value,chemical reaction, bio-chemical reaction, bio-electro-chemical reaction,reaction speed, reaction energy, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, or bondingstrength; the physical property is density or geometric size; theacoustic property is frequency, speed of acoustic waves, acousticfrequency and intensity spectrum distribution, acoustic intensity,acoustical absorption, or acoustical resonance; and the mechanicalproperty is internal pressure, hardness, shear strength, elongationstrength, fracture stress, adhesion, mechanical resonance frequency,elasticity, plasticity, or compressibility.

In some embodiments, the data recorded by the first detectingmicro-device is filtered by a phase lock-in technology to remove noiseunsynchronized to the data recorded by the first probing micro-device orthe clock micro-device. The filtered data may have a higher signal tonoise data ratio.

Another innovative aspect of the present invention is the use ofmicro-devices for obtaining real time data and information at thecellular structure level, such as using a micro voltage comparator,four-point probe and other circuitry designs to measure cell surface orbulk electrical properties including resting potential and surfacecharge for differentiating normal cells and cancer cells. The cellsurface charge differentiation can be an important factor in decidingthe healthy or unhealthy status of a cell and, accordingly, the propertreatment thereof.

For example, in a time of flight approach to obtain dynamic informationon a biological subject (e.g., a cell, a substructure of a cell, a DNAor RNA molecule, or a virus), a first micro-device is first used to senda signal to perturb the biological subject to be diagnosed, and then asecond micro-device is employed to accurately measure the response fromthe biological subject. In one arrangement, the first micro-device andthe second device are positioned at a desired distance L apart, with abiological subject to be measured flowing from the first micro-devicetowards the second micro-device. When the biological subject samplepasses the first micro-device, the micro-device sends a signal to thepassing biological sample, and then the second micro-device detects theresponse or retention of the perturbation signal on the entity. From thedistance between the two micro-devices, time interval, the nature ofperturbation by the first micro-device, and measured changes on thebiological subject during the time of flight, microscopic and dynamicproperties of the biological subject can be measured and data obtained.In another arrangement, a first micro-device is used to probe thebiological subject by first applying a signal (such as a charge) andthen detecting the response from the biological subject with a secondmicro-device as a function of time.

Another novel area of this application is the invention ofmicro-indentation probes and micro-probes for measuring a range ofphysical properties (such as mechanical properties) of biologicalsubjects. Examples of such physical properties include but not limitedto hardness, shear strength, elongation strength, fracture stress, andproperties related to cell membranes as the membranes may be a criticalcomponent in disease diagnosis.

Still yet another aspect of this invention is the design, fabrication,and integration of the various components in the disease detectionapparatus. These components include, e.g., a sample containment anddelivery unit; an array of sample delivery channels; a central diseasedetection unit comprising multiple detection probes, a central controlunit comprising a logic processing unit, a memory unit, a sensor, asignal transmitter, a signal receiver, and an application specific chip;and a waste sample treatment unit in which used sample can be treated,recycled, processed for reuse, or disposed.

Another key novel aspect of the current application is the design,integration, and fabrication process flow of micro-devices capable ofmaking highly sensitive and advanced measurements on very weak signalsin biological systems for disease detection under complicatedenvironment with very weak signal and relatively high noise background.Those novel capabilities using the class of micro-devices disclosed inthis invention for disease detection include, e.g., making dynamicmeasurements, real time measurements (such as time of flightmeasurements, and combination of using probe signal and detectingresponse signal), phase lock-in technique to reduce background noise,and 4-point probe techniques to measure very weak signals, and uniqueand novel probes to measure various electronic, electromagnetic andmagnetic properties of biological samples at the single cell, biologicalsubject (e.g., virus) or molecule (e.g., DNA or RNA) level.

Finally, another aspect of this invention relates to apparatus fordetecting disease in a biological subject. The apparatus includes adetection device fabricated by a method comprising: providing asubstrate; sequentially depositing a first material and a secondmaterial as two layers onto the substrate to form a material stack;patterning the second material by microelectronic processes to form afirst desired feature; depositing a third material onto the materialstack to cover the second material; optionally patterning the first andthird materials by microelectronic processes to form a second desiredfeature; and optionally depositing a fourth material onto the materialstack. The first material and third material can be the same ordifferent. The detection device is capable of probing the biologicalsubject to be detected and giving rise to a response signal.

In some embodiments, the fabrication method further comprises cappingthe top of the material stack to form an enclosed trench.

In some other embodiments, the capping comprises sealing or capping thetop of the material stack with an imaging device onto the materialstack.

In still some other embodiments, the apparatus further includes apre-processing unit (chambers) for pre-screening and enhancing adiseased biological subject for further testing, channels for carryingfluidic sample to flow through, probes for probing and disturbing thebiological subject being tested for generating response signals,detection probes for measuring properties and response signals of thebiological subject, or an imaging device for observing and recordingproperties and behaviors of the biological subject.

In yet some other embodiments, the detection device has typical channeldimensions ranging from about 2 microns×2 microns to about 100microns×100 microns in cross sectional area for a square-shaped channel,a radius ranging from about 1 micron to about 20 microns in crosssectional area for a circular shaped channel, and a typical probedimension ranging from about 0.5 micron×0.5 micron to about 20microns×20 microns in cross sectional area for a square-shaped probe.Alternatively, the detection device has typical channel dimensionsranging from about 6 microns x 6 microns to about 14 microns×14 micronsin cross sectional area for a square-shaped channel, a radius rangingfrom about 3 microns to about 8 microns in cross sectional area for acircular shaped channel, and a typical probe dimension ranging fromabout 0.5 micron×0.5 micron to about 10 microns×10 microns in crosssectional area for a square shaped probe.

In yet still some embodiments, the first and the fourth materials eachcomprise un-doped oxide (SiO₂), doped oxide, silicon nitride, a polymermaterial, glass, or an electrically insulating material; the second andthird materials each comprise an electrically conductive material,aluminum, an aluminum alloy, copper, a copper alloy, tungsten, atungsten alloy, gold, a gold alloy, silver, a silver alloy, an opticalmaterial, an thermal sensitive material, a magnetic material, a pressuresensitive material, a mechanical stress sensitive material, an ionemission sensitive material, and a piezo-electric material.

In yet still some other embodiments, where the second and fourthmaterials can be fabricated at the same level as detectors, or as probesand detectors, the first and the third materials each comprise un-dopedoxide (SiO₂), doped oxide, silicon nitride, a polymer material, glass,or an electrically insulating material; the second and fourth materialseach comprise an electrically conductive material (e.g., aluminum, analuminum alloy, copper, a copper alloy, tungsten, a tungsten alloy,gold, a gold alloy, silver, or a silver alloy), an optical material(e.g., anisotropic optical material, glass, glass-ceramic, laser gainmedia, nonlinear optical material, phosphor and scintillator,transparent material), an thermal sensitive material, a magneticmaterial, a pressure sensitive material, a mechanical stress sensitivematerial, an ion emission sensitive material, and a piezo-electricmaterial (e.g., quartz, berlinite, gallium, orthophosphate, GaPO₄,tourmaline, ceramics, barium, titanate, BatiO₃, lead zirconate, titanatePZT, zinc oxide, aluminum nitride, and a polyvinylidene fluoride).

In further embodiments, the detection device comprises at least oneprobe, at least one detector, at least one pair of probe and detector inwhich the probe generates a probing or disturbing signal onto thebiological subject to give a response signal and the detector measuresthe response signal thus generated.

In other aspects, the present invention provides methods for fabricatingmicro-devices or micro-detectors of this invention by microelectronicprocess which may include deposition, lithography, etch, cleaning,direct writing, molecular self assembly, laser oblation, electron beamwriting, x-ray writing, diffusion, ion implantation, cleaning,polishing, planarization, or packaging.

In some embodiments, the methods fabricating a micro-device ormicro-detector include depositing various materials on a substrate and,in the interims of depositing every two materials, patterning some orall of the deposited materials by a microelectronic process. Themicro-device or micro-detector thus fabricated is capable of measuringat the microscopic level an electric, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-physical, bio-physical-chemical, physical-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property of abiologic subject with which the micro-device or micro-detector is tocontact.

The electrical property may include surface charge, surface potential,resting potential, action potential, electrical voltage, electricalcurrent, electrical field distribution, electrical charge distribution,electric dipole, electric quadruple, three-dimensional electrical orcharge cloud distribution, electrical properties at telomere of DNA andchromosome, dynamic changes in electrical properties, dynamic changes inpotential, dynamic changes in surface charge, dynamic changes incurrent, dynamic changes in electrical field, dynamic changes inelectrical voltage, dynamic changes in electrical distribution, dynamicchanges in electronic cloud distribution, or impedance; the thermalproperty may include temperature, or vibrational frequency of biologicalitem or molecules; the optical property may include optical absorption,optical transmission, optical reflection, optical-electrical property,brightness, or fluorescent emission; the chemical property may includepH value, chemical reaction, bio-chemical reaction, bio-electro-chemicalreaction, reaction speed, reaction energy, speed of reaction, oxygenconcentration, oxygen consumption rate, ionic strength, catalyticbehavior, or bonding strength; the physical property may include densityor geometric size; the acoustic property may include frequency, speed ofacoustic waves, acoustic frequency and intensity spectrum distribution,acoustic intensity, acoustical absorption, or acoustical resonance; andthe mechanical property may include internal pressure, hardness, shearstrength, elongation strength, fracture stress, adhesion, mechanicalresonance frequency, elasticity, plasticity, or compressibility.

In some other embodiments, the fabrication methods each include thesteps of:

-   -   providing a substrate;    -   depositing a first material onto the substrate;    -   depositing a second material onto the first material and then        patterning the second material by a microelectronic process; and    -   repeating the second step at least once with a material that can        be the same as or different from any of the previously deposited        materials.

The methods may further include removal of a stack of multiple layers ofmaterials by wet etch, dry etch, or vapor etch.

In these methods, the materials used in the repeated steps can be thesame as or different from the first or second material. At least one ofthe materials used in fabricating the micro-device is a biologicalmaterial, a polymer, a piezo-electric material, a semiconductormaterial, an electrically insulating material, or an electricallyconductive material.

The micro-device thus fabricated can have one or more characters orfunctions of the following: moving in any direction; being capable ofsorting, probing, measuring, communicating, or modifying a biologicalsubject.

Still, the methods may further include one or more of the followingsteps:

-   -   depositing a third material on the second material and then        patterning the third material by a planarization process;    -   depositing a fourth material on the third material and        patterning the fourth material by microelectronic processes;    -   patterning the third material using a microelectronic process        with the fourth material serving as a hardmask;    -   coupling two devices that are thus fabricated and symmetric to        form a detecting device with channels or to form a probing        device capable to sending a signal to a biological subject and        result in a response;    -   integrating three or more micro-devices to give an enhanced        device with an array of the channels.

Still further, the methods may include the steps of:

-   -   before depositing the second material, patterning the first        material by a microelectronic process to give rise to at least        one patterned residual and leaving part of the substrate surface        uncovered by the first material;    -   creating an opening in the second material to expose part of the        patterned residual of the first material; and    -   filling up the opening in the second material with a third        material; wherein the second material is a non-electrically        conductive material.

The micro-device thus obtained may include a micro-trench (or channel)having side-walls and a probe embedded in the micro-trench or channel'ssidewalls. Each channel's entrance may be optionally bell-mouthed; theshape of each channel's cross-section is rectangle, ellipse, circle, orpolygon. The dimension of the micro-trench may range from about 0.1 umto about 500 um.

The micro-trench of the micro-device can be capped with a flat panel orcoupling two micro-trenches to form one or more channels. The flat panelmay comprise silicon, SiGe, SiO₂, Al₂O₃, acrylate polymer, AgInSbTe,synthetic alexandrite, arsenic triselenide, arsenic trisulfide, bariumfluoride, CR-39, cadmium selenide, caesium cadmium chloride, calcite,calcium fluoride, chalcogenide glass, gallium phosphide, GeSbTe,germanium, germanium dioxide, glass code, hydrogen silsesquioxane,Iceland spar, liquid crystal, lithium fluoride, lumicera, METATOY,magnesium fluoride, magnesium oxide, negative index metamaterials,neutron super mirror, phosphor, picarin, poly(methyl methacrylate),polycarbonate, potassium bromide, sapphire, scotophor, spectralon,speculum metal, split-ring resonator, strontium fluoride, yttriumaluminum garnet, yttrium lithium fluoride, yttrium orthovanadate, ZBLAN,zinc selenide, or zinc sulfide.

In some other embodiments, the methods for fabricating a micro-device ormicro-detector of this invention include the steps of:

-   -   providing a substrate;    -   sequentially depositing a first material and a second material        as two layers onto the substrate to form a material stack;    -   patterning the second material by microelectronic processes to        form a first desired feature; depositing a third material onto        the material stack to cover the second material and optionally        the first material;    -   optionally patterning the first and third materials by        microelectronic processes to form a second desired feature; and    -   optionally depositing a fourth material onto the material stack.

They may further include:

-   -   fabricating at least an additional component onto the substrate        before sequentially depositing the first material and the second        material as layers onto the substrate, wherein the additional        component comprises a data storage component, a signal        processing component, a memory storage component, a signal        receiver, a signal transmitting component, a logic processing        component, or an RF component; or    -   fabricating at least an integrated circuit onto the substrate        before sequentially depositing the first material and the second        material as layers onto the substrate, wherein the integrated        circuit comprises a data storage circuit, a signal processing        circuit, a memory storage circuit, a signal transmitting        circuit, a sensor, or a logic processing circuit.

In some instances, the first material and the third material are thesame; the first material and the third material are electricallyinsulating (e.g., an oxide, doped oxide, silicon nitride, or a polymer);the first material and the fourth material are the same; the firstmaterial and the fourth material are electronically insulating; thesecond material or the third material is an electrical conductivematerial, a magnetic material, an electro-magnetic material, an opticalmaterial, a thermal sensitive material, a pressure sensitive material,an ion emission sensitive material, or a piezo-electric material.

In some other instances, the second material is an electricallyconductive material, a piezo-electric material, a semiconductormaterial, a thermal sensitive material, a magnetic material, a pressuresensitive material, a mechanical stress sensitive material, an ionemission sensitive material, an optical material, or a combinationthereof. For example, it may include copper, aluminum, tungsten, gold,silver, the alloys thereof, or glass.

The detector thus fabricated may be capable of probing or disturbing abiological subject to be measured; and it may have a recessed form, or atrench form in the layers of the third and first materials. In thedetector, the second material may be aligned with the wall of the trenchform in the layers of the third and first materials.

In some instances, the methods may further include the step of cappingthe top of the material stack to cover the third material and form anenclosed trench. As an example, the capping may include sealing orcapping the top of the material stack with a layer of material, animaging device, a camera, a viewing station, an acoustic detector, athermal detector, an ion emission detector, or a thermal recorder ontothe material stack.

In some other instances, the methods may still further include one ormore of the following steps:

-   -   fabricating at least one integrated circuit onto the substrate        before sequentially depositing the first material and the second        material as layers onto the substrate, wherein the circuit        comprises a data storage circuit, a signal processing circuit, a        memory storage circuit, a sensor, a signal transmitting circuit,        a sensor, or a logic processing circuit;    -   planarizing the third material using a chemical mechanical        polishing process or an etch back process after depositing the        third material onto the material stack and before patterning the        first and the third materials;    -   planarizing the third material using a chemical mechanical        polishing process or an etch back process to form a detector        capable of detecting a response signal from the biological        subject;    -   patterning the fourth material to form a hole at a selected        location after depositing the fourth material onto the material        stack;    -   removing the third material from the material stack by wet or        vapor etch to form a detection chamber between the fourth        material and the substrate;    -   removing the first material from the material stack by wet or        vapor etch to form a channel; capping the top of the material        stack to form an enclosed trench or channel;    -   sealing or capping the top of the material stack with a fifth        material to form an enclosed channel capable of observing and        recording the biological subject; or    -   sealing or capping the top of the material stack with an imaging        device, a detector, an optical sensor, a camera, a viewing        station, an acoustic detector, a thermal detector, an electrical        detector, an ion emission detector, or a thermal recorder onto        the material stack.

In still some embodiments, the methods for fabricating a micro-device ofthis invention include the steps of:

-   -   providing a substrate;    -   sequentially depositing a first material and a second material        as layers onto the substrate to form a material stack;    -   patterning the second material by microelectronic processes to        form at least a portion of a recessed area in the second        material (e.g., to form a probe, a detector or an integrated        unit with sub-component for detection);    -   depositing a third material onto the material stack to cover the        second material, and removing the portion of the third material        above the second material by etch back or polishing process;    -   patterning the third material by lithography and etch processes        to remove at least a portion of the third material;    -   depositing a fourth material onto the material stack to cover        the second and third material, and removing the portion of the        fourth material above the second and third material by etch back        or polishing process; and    -   optionally, depositing a fifth material and repeating the above        process sequence used for the third material.

In some instances, they may further include one or more steps of thefollowing:

-   -   fabricating at least an additional component onto the substrate        before sequentially depositing the first material and the second        material as layers onto the substrate, wherein the additional        component comprises a data storage component, a signal        processing component, a memory storage component, a signal        transmitting component, a logic processing component, or an RF        component; and    -   fabricating at least one integrated circuit onto the substrate        before sequentially depositing the first material and the second        material as layers onto the substrate, wherein the integrated        circuit comprises a data storage circuit, a signal processing        circuit, a memory storage circuit, a signal transmitting        circuit, a sensor, or a logic processing circuit.

The substrate can be silicon, polysilicon, silicon nitride, or polymermaterial; the first material is oxide, doped oxide, silicon nitride, orpolymer material. The second and the fourth materials can be the same(e.g., both being an electrical conductive material, semiconductormaterial, piezo-electric material, thermal sensitive material, an ionemission sensitive material, a magnetic material, a pressure sensitivematerial, a mechanical stress sensitive material, or optical material).Specific examples of suitable materials include aluminum, copper,tungsten, gold, silver, the alloys thereof, quartz, berlinite, gallium,orthophosphate, GaPO₄, tourmalines, ceramics, barium, titanate, BatiO₃,lead zirconate, titanate PZT, zinc oxide, aluminum nitride, andpolyvinylidene fluoride.

In still a further aspect, the invention provides piezo-electricmicro-detectors. Each of these micro-detectors comprises a substrate, apiezo-electric material, an electronically conductive material, amaterial that is neither piezo-electric nor electronically conductive,wherein the piezo-electric material is placed between the electronicallyconductive material and the material that is neither piezo-electric norelectronically conductive, and the material that is neitherpiezo-electric nor electronically conductive is placed between thesubstrate and the piezo-electric material, wherein the micro-detector iscapable of detecting, at the microscopic level, a property of an objectto be detected.

In some embodiments, a portion of the piezo-electric material isprojecting out of the other part of the micro-detector and is notsupported or surrounded by the other materials in the micro-detector.The projecting piezo-electric material can be, e.g., in the shape of alayer or a stick and can have a minimum length of one angstrom.

In some embodiments, the projecting piezo-electric material has an axelthat is essentially parallel to the surface of the substrate.

The projecting piezo-electric material is capable of detecting, at themicroscopic level, a property of the object to be detected. The propertycan be an electric, magnetic, electromagnetic, thermal, optical,acoustical, biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property of theobject to be detected. For example, the electrical property can besurface charge, surface potential, resting potential, electricalcurrent, electrical field distribution, electric dipole, electricquadruple, three-dimensional electrical or charge cloud distribution,electrical properties at telomere of DNA and chromosome, or impedance;the thermal property can be temperature, or vibrational frequency ofbiological item or molecules; the optical property can be opticalabsorption, optical transmission, optical reflection, optical-electricalproperty, brightness, or fluorescent emission; the chemical property canbe pH value, chemical reaction, bio-chemical reaction,bio-electro-chemical reaction, reaction speed, reaction energy, oxygenconcentration, oxygen consumption rate, ionic strength, catalyticbehavior, or bonding strength; the physical property can be density orgeometric size; the acoustic property can be frequency, speed ofacoustic waves, acoustic frequency and intensity spectrum distribution,acoustic intensity, acoustical absorption, or acoustical resonance; andthe mechanical property can be internal pressure, hardness, shearstrength, elongation strength, fracture stress, adhesion, mechanicalresonance frequency, elasticity, plasticity, or compressibility.

In some embodiments, the electronically conductive material is connectedto the piezo-electric material and capable of delivery signal from thepiezo-electric material to a measuring or recording device.

In some embodiments, the piezo-electric material expands when it detectsan electric property from the object to be tested, or the piezo-electricmaterial gives rise to an electric currency when it detects a mechanicalstress.

The piezo-electric material comprises a crystal, a ceramics, zinc oxide,aluminum nitride, polyvinylidene fluoride, lithium tantalite, lanthanumgallium silicate, or potassium sodium tartrate. Examples of suitablecrystals include tourmaline, tourmaline, topaz, quartz, Rochelle salt,Berlinite, and gallium orthophosphate; while examples of suitableceramics include BaTiO₃, KNbO₃, Ba₂NaNb₅O₅, LiNbO₃, SrTiO₃, Pb(ZrTi)O₃,Pb₂KNb₅O₁₅, LiTaO₃, BiFeO₃, and NaxWO₃.

In some embodiments, the electronically conductive material comprises anelectric conductor or semiconductor. The electric conductor may includea metal or graphite, and the semiconductor may include a crystal or aceramics.

In some embodiments, the material that is neither piezo-electric norelectronically conductive, is a wet etching stop material.

The piezo-electric micro-detectors described above can be fabricated bya process comprising microelectronics technologies. Accordingly, theinvention further provides methods for fabricating a piezo-electricmicro-detector. Each method includes the following steps:

-   -   providing a substrate;    -   depositing a first material onto the substrate;    -   optionally planarizing the first material;    -   depositing a second material onto the optionally planarized        first material; wherein the second material is neither        piezo-electric nor electrically conductive;    -   patterning the second material to create at least one recessed        area in the second material;    -   depositing a third, piezo-electric material on the second        material to fill its recessed area in the second material and        cover the second material;    -   patterning the third, piezo-electric material to create at least        one recessed area in the piezo-electric material;    -   depositing a fourth material onto the third, piezo-electric        material to fill its recessed area and optionally to cover the        third, piezo-electric material; wherein the fourth material can        be the same as or different from the second material, and the        fourth material is neither piezo-electric nor electrically        conductive;    -   optionally patterning the fourth material to give it a certain        configuration;    -   optionally depositing a fifth material onto the optionally        patterned fourth material, wherein the fifth material can be the        same as or different from the second material, the fifth        material is different from the fourth material, and the fifth        material is neither piezo-electric nor electrically conductive;    -   patterning the fourth material and optional fifth material to        create an opening that exposes the third, piezo-electric        material;    -   depositing a sixth, electrically conductive material to fill the        opening in the fourth material and optional fifth material, and        optionally covering part of the fifth material; and    -   patterning all the materials above the substrate to expose all        the materials, and    -   patterning the second and fourth materials sandwiching the        piezo-electric material to expose at least part of the        piezo-electric material.

If desired, additional material layers (e.g., seventh material layer, orseventh and eighth material layers) can be deposited, patterned,cleaned, or planarized to form additional structures with more features,functionalities, and complexities.

In some embodiments, the second material or the fifth material is a wetetch stop material.

In some embodiments, the patterning process comprises lithography andetching.

In some embodiments, a portion of the patterned piezo-electric materialis projecting from the other material(s) with which it is connected, andthe projecting piezo-electric material is in the shape of a layer or astick. For example, the projecting piezo-electric material has an axelthat is essentially parallel to the surface of the substrate.

Yet in another aspect, the invention provides methods for detecting, atthe microscopic level, a mechanical or electric property of a biologicalsubject. Each method includes the steps of: providing a piezo-electricmicro-detector comprising a substrate, a piezo-electric material, anelectronically conductive material, a material that is neitherpiezo-electric nor electronically conductive, wherein the piezo-electricmaterial is placed between the electronically conductive material andthe material that is neither piezo-electric nor electronicallyconductive, and the material that is neither piezo-electric norelectronically conductive is placed between the substrate and thepiezo-electric material; contacting the piezo-electric micro-detectorwith the biological subject to be detected, wherein the piezo-electricmicro-detector detects the mechanical or electric property of thebiological subject upon the contact and converts the mechanical orelectric property to generate an electric or mechanical property, andtransferring the electric or mechanical property thus generated throughthe electrically conductive material to a recording device.

As used herein, the term “or” is meant to include both “and” and “or”.It may be interchanged with “and/or.”

As used herein, a singular noun is meant to include its plural meaning.For instance, a micro device can mean either a single micro device ormultiple micro-devices.

As used herein, the term “patterning” means shaping a material into acertain physical form or pattern, including a plane (in which case“patterning” would also mean “planarization.”)

As used herein, the term “a biological subject” or “a biological sample”for analysis or test or diagnosis refers to the subject to be analyzedby a disease detection apparatus. It can be a single cell, a singlebiological molecular (e.g., DNA, RNA, or protein), a single biologicalsubject (e.g., a single cell or virus), any other sufficiently smallunit or fundamental biological composition, or a sample of a subject'sorgan or tissue that may having a disease or disorder.

As used herein, the term “disease” is interchangeable with the term“disorder” and generally refers to any abnormal microscopic property orcondition (e.g., a physical condition) of a biological subject (e.g., amammal or biological species).

As used herein, the term “subject” generally refers to a mammal, e.g., ahuman person.

As used herein, the term “microscopic level” refers to the subject beinganalyzed by the disease detection apparatus of this invention is of amicroscopic nature and can be a single cell, a single biologicalmolecular (e.g., DNA, RNA, or protein), a single biological subject(e.g., a single cell or virus), and other sufficiently small unit orfundamental biological composition.

As used herein, a “micro-device” or “micro device” can be any of a widerange of materials, properties, shapes, and degree of complexity andintegration. The term has a general meaning for an application from asingle material to a very complex device comprising multiple materialswith multiple sub units and multiple functions. The complexitycontemplated in the present invention ranges from a very small, singleparticle with a set of desired properties to a fairly complicated,integrated unit with various functional units contained therein. Forexample, a simple micro-device could be a single spherical article ofmanufacture of a diameter as small as 100 angstroms with a desiredhardness, a desired surface charge, or a desired organic chemistryabsorbed on its surface. A more complex micro device could be a 1millimeter device with a sensor, a simple calculator, a memory unit, alogic unit, and a cutter all integrated onto it. In the former case, theparticle can be formed via a fumed or colloidal precipitation process,while the device with various components integrated onto it can befabricated using various integrated circuit manufacturing processes.

A micro device used in the present invention can range in size (e.g.,diameter) from on the order of about 1 angstrom to on the order of about5 millimeters. For instance, a micro-device ranging in size from on theorder of about 10 angstroms to on the order of 100 microns can be usedin this invention for targeting biological molecules, entities orcompositions of small sizes such as cell structures, DNA, and bacteria.Or, a micro-device ranging in size from on the order of about one micronto the order of about 5 millimeters can be used in the present inventionfor targeting relatively large biological matters such as a portion of ahuman organ. As an example, a simple micro device defined in the presentapplication can be a single particle of a diameter less than 100angstroms, with desired surface properties (e.g., with surface charge ora chemical coating) for preferential absorption or adsorption into atargeted type of cell.

The present invention further provides an apparatus for detecting adisease in a biological subject, which comprises a pre-processing unit,a probing and detecting unit, a signal processing unit, and a disposalprocessing unit.

In some embodiments of the apparatus, the pre-processing unit includes asample filtration unit, a recharging unit, a constant pressure deliveryunit, and a sample pre-probing disturbing unit. This increases thecontraction ratio of certain substance of interests (such as cancercells) and therefore makes the apparatus more effective and efficient indetecting the targeted biological subject (such as cancer cells).

In some embodiments, the filtration unit can filter off unwantedsubstance by physical filtration (e.g., based on the electronic chargeor size of the substance) or separation by chemical reaction (therebycompletely removing the undesirable substances), biochemical reaction,electro-mechanical reaction, electro-chemical reaction, or biologicalreaction.

In some embodiments, the sample filtration unit can include an entrancechannel, a disturbing fluid channel, an accelerating chamber, and aslit. The slit and the interior walls of the entrance channel define twochannels (e.g., a top channel and a bottom channel) wherein thebiological subject can be separated due to the differences in itsproperty (e.g., electric or physical property).

In some embodiments, a bio-compatible fluid can be injected into thedisturbing fluid channel to separate the biological subject. Forexample, the bio-compatible fluid can be injected from the entrance ofthe disturbing fluid channel and deliver to an opening in the entrancechannel wall. The bio-compatible fluid can be liquid or semi-liquid, andcan include saline, water, plasma, an oxygen-rich liquid, or anycombination thereof.

In some other embodiments, the angle between the entrance channel andthe disturbing fluid channel ranges from about 0° to about 180° (e.g.,from about 30° to about 150°, from about 60° to about 120°, or fromabout 75° to about 105°, or about 90°).

In some other embodiments, the width of each channel can range fromabout 1 nm to about 1 mm (e.g., from about 2 nm to about 0.6 mm or fromabout 10 nm to about 0.2 mm).

In some other embodiments, at least one of the channels comprises oneprobing device attached to the channel's sidewall, and the probingdevice is capable of measuring at the microscopic level an electric,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject. Examples of the electrical property include surfacecharge, surface potential, resting potential, electrical current,electrical field distribution, electric dipole, electric quadruple,three-dimensional electrical or charge cloud distribution, electricalproperties at telomere of DNA and chromosome, and impedance. Examples ofthe thermal property include temperature and vibrational frequency.Examples of the optical property include optical absorption, opticaltransmission, optical reflection, optical-electrical property,brightness, and fluorescent emission. Examples of the chemical propertyinclude pH value, chemical reaction, bio-chemical reaction,bio-electro-chemical reaction, reaction speed, reaction energy, speed ofreaction, oxygen concentration, oxygen consumption rate, ionic strength,catalytic behavior, and bonding strength. Examples of the physicalproperty include density and geometric size. Examples of the acousticproperty include frequency, speed of acoustic waves, acoustic frequencyand intensity spectrum distribution, acoustic intensity, acousticalabsorption, and acoustical resonance. Examples of the mechanicalproperty include internal pressure, hardness, shear strength, elongationstrength, fracture stress, adhesion, mechanical resonance frequency,elasticity, plasticity, and compressibility.

In some embodiments, at least one of the channels comprises at least twoprobing devices attached to the channel's sidewalls, and the probingdevices are capable of measuring at the microscopic level an electric,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject. The probing devices measure the same or differentproperties at the same time or different times.

The two or more probing devices can be placed with a desired distancebetween each other (at least 10 angstroms). Examples of the desireddistance include from about 10 nm to about 100 mm, from about 100 nm toabout 10 mm, from about 1 mm to about 10 mm.

In some embodiments, the micro-device of this invention comprises atleast one probe and at least one detector. The probe can be utilized tolaunch a probing signal to probe the biological subject, and thedetector can detect the biological subject's response (signal) to theprobing signal. As an example, a micro-device with at least one acousticprobe (such as an acoustic transducer or microphone) and at least onedetector (such as an acoustic signal receiver) is utilized forbiological subject detection, wherein the acoustic probe and detectormay be constructed with, among others, one or more piezo-electricmaterials. In this example, an acoustic signal is first launched, andscanned across its frequency range (e.g., from sub Hz to over MHz) bythe probe. The response signal to the launched acoustic signal by theprobe is then collected by the detector, and subsequently recorded,amplified (e.g., by a lock-in amplifier), and analyzed. The responsesignal contains characteristic information of a biological subject thatis tested. For example, depending on certain properties of the testedbiological subject, the detected acoustic resonant frequency, intensity,frequency versus intensity spectrum, or intensity distribution by thedetector may indicate characteristic information about the testedbiological subject. Such information includes density, densitydistribution, absorption properties, shape, surface properties, andother static and dynamic properties of the biological subject.

In some embodiments, the sample filtration unit can include an entrancechannel, a biocompatible filter, an exit channel, or any combinationthereof. When a biological subject passes through the entrance channeltoward the exit channel, the biological subject of a size larger thanthe filter hole will be blocked against the exit channel, resulting inthe smaller biological subject being flushed out through the exitchannel. A biocompatible fluid is injected from the exit to carry thebiological subject accumulated around the filter and flush out from thechannel. The biological subject with a large size is then filtered forfurther analysis and detection in the detecting component or unit of theapparatus.

In some embodiments, the sample pre-probing disturbing unit can includeone micro-device with a channel, a slit located inside the channel, andoptionally two plates outside the channel. The two plates can apply asignal, e.g., an electronic voltage, to the biological subject travelingthrough the channel and separates it based on the electronic charge thebiological subject carries. The slit and the interior channels of thechannel define two channels where the separated biological subjectsenter and optionally are detected for its property at the microscopiclevel.

In some embodiments, the sample pre-probing disturbing unit applies tothe biological subject an electric, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical signal. Thesignal can be applied, e.g., with the two plates described above or inother means (depending on the nature of the signal). The signal asapplied can be pulsed or constant.

In some embodiments, the recharging unit recharges nutrient or respiringgas (such as oxygen) to the biological subject. Alternatively, it canalso clean up the metabolite of the biological subject. With such arecharging unit, the life stability of the biological subject in thesample is sustained and its use is extended, thereby giving moreaccurate and reliable detecting results. Examples of nutrient includebiocompatible strong or weak electrolyte, amino acid, mineral, ions,oxygen, oxygen-rich liquid, intravenous drip, glucose, and protein.Another example of the nutrient is a solution containing nano-particlesthat can be selectively absorbed by certain biological subjects (e.g.,cells or viruses).

The recharging system can be separate from and outside of the othercomponents of the apparatus. Alternatively, it can also be installedwithin one of the other components, e.g., the probing and detecting unitor the disposal processing unit.

In some other embodiments, the signal processing unit comprises anamplifier (e.g., a lock-in amplifier), an A/D (alternate/direct electriccurrent or analog to digital) converter, a micro-computer, amanipulator, a display, and network connections.

In some instance, the signal processing unit collects more than onesignal (i.e., multiple signals), and the multiple signals can beintegrated to cancel out noise or to enhance the signal to noise ratio.The multiple signals can be signals from multiple locations or frommultiple times.

Biological subjects that can be detected by the apparatus include, e.g.,blood, urine, saliva, tear, and sweat. The detection results canindicate the possible occurrence or presence of a disease (e.g., one inits early stage) in the biological subject.

As used herein, the term “absorption” typically means a physical bondingbetween the surface and the material attached to it (absorbed onto it,in this case). On the other hand, the word “adsorption” generally meansa stronger, chemical bonding between the two. These properties are veryimportant for the present invention as they can be effectively used fortargeted attachment by desired micro devices for measurement at themicroscopic level.

As used herein, the term “contact” (as in “the first micro-devicecontacts a biologic entity”) is meant to include both “direct” (orphysical) contact and “non-direct” (or indirect or non-physical)contact. When two subjects are in “direct” contact, there is generallyno measurable space or distance between the contact points of these twosubjects; whereas when they are in “indirect” contact, there is ameasurable space or distance between the contact points of these twosubjects.

As used herein, the term “probe” or “probing,” in addition to itsdictionary meaning, could mean applying a signal (e.g., an electrical,acoustic, magnetic or thermal signal) to a subject and therebystimulating the subject and causing it to have some kind of intrinsicresponse.

As used herein, the term “electric property” refers to surface charge,surface potential, electrical field, charge distribution, electricalfield distribution, resting potential, action potential, or impedance ofa biological subject to be analyzed.

As used herein, the term “magnetic property” refers to diamagnetic,paramagnetic, or ferromagnetic.

As used herein, the term “electromagnetic property” refers to propertythat has both electric and magnetic dimensions.

As used herein, the term “thermal property” refers to temperature,freezing point, melting point, evaporation temperature, glass transitiontemperature, or thermal conductivity.

As used herein, the term “optical property” refers to reflection,optical absorption, optical scattering, wave length dependentproperties, color, luster, brilliance, scintillation, or dispersion.

As used herein, the term “acoustical property” refers to thecharacteristics found within a structure that determine the quality ofsound in its relevance to hearing. It can generally be measured by theacoustic absorption coefficient. See, e.g., U.S. Pat. No. 3,915,016, formeans and methods for determining an acoustical property of a material;T. J. Cox et al., Acoustic Absorbers and Diffusers, 2004, Spon Press.

As used herein, the term “biological property” is meant to generallyinclude chemical and physical properties of a biological subject.

As used herein, the term “chemical property” refers to pH value, ionicstrength, or bonding strength within the biological sample.

As used herein, the term “physical property” refers to any measurableproperty the value of which describes a physical system's state at anygiven moment in time. The physical properties of a biological sample mayinclude, but are not limited to absorption, albedo, area, brittleness,boiling point, capacitance, color, concentration, density, dielectric,electric charge, electrical conductivity, electrical impedance, electricfield, electric potential, emission, flow rate, fluidity, frequency,inductance, intrinsic impedance, intensity, irradiance, luminance,luster, malleability, magnetic field, magnetic flux, mass, meltingpoint, momentum, permeability, permittivity, pressure, radiance,solubility, specific heat, strength, temperature, tension, thermalconductivity, velocity, viscosity, volume, and wave impedance.

As used herein, the term “mechanical property” refers to strength,hardness, toughness, elasticity, plasticity, brittleness, ductility,shear strength, elongation strength, fracture stress, or adhesion of thebiological sample.

As used herein, the term “conductive material” (or its equivalent“electric conductor”) is a material which contains movable electriccharges. A conductive material can be a metal (e.g., copper, silver, orgold) or non-metallic (e.g., graphite, solutions of salts, plasmas, orconductive polymers). In metallic conductors, such as copper oraluminum, the movable charged particles are electrons (see electricalconduction). Positive charges may also be mobile in the form of atoms ina lattice that are missing electrons (known as holes), or in the form ofions, such as in the electrolyte of a battery.

As used herein, the term “electrically insulating material” (also knownas “insulator” or “dielectric”) refers to a material that resists theflow of electric current. An insulating material has atoms with tightlybonded valence electrons. Examples of electrically insulating materialsinclude glass or organic polymers (e.g., rubber, plastics, or Teflon).

As used herein, the term “semiconductor” (also known as “semiconductingmaterial”) refers to a material with electrical conductivity due toelectron flow (as opposed to ionic conductivity) intermediate inmagnitude between that of a conductor and an insulator. Examples ofinorganic semiconductors include silicon, silicon-based materials, andgermanium. Examples of organic semiconductors include such aromatichydrocarbons as the polycyclic aromatic compounds pentacene, anthracene,and rubrene; and polymeric organic semiconductors such aspoly(3-hexylthiophene), poly(p-phenylene vinylene), polyacetylene andits derivatives. Semiconducting materials can be crystalline solids(e.g., silicon), amorphous (e.g., hydrogenated amorphous silicon andmixtures of arsenic, selenium and tellurium in a variety ofproportions), or even liquid.

As used herein, the term “biological material” has the same meaning of“biomaterial” as understood by a person skilled in the art. Withoutlimiting its meaning, biological materials or biomaterials can generallybe produced either in nature or synthesized in the laboratory using avariety of chemical approaches utilizing organic compounds (e.g., smallorganic molecules or polymers) or inorganic compounds (e.g., metalliccomponents or ceramics). They generally can be used or adapted for amedical application, and thus comprise whole or part of a livingstructure or biomedical device which performs, augments, or replaces anatural function. Such functions may be benign, like being used for aheart valve, or may be bioactive with a more interactive functionalitysuch as hydroxyl-apatite coated hip implants. Biomaterials can also beused every day in dental applications, surgery, and drug delivery. Forinstance, a construct with impregnated pharmaceutical products can beplaced into the body, which permits the prolonged release of a drug overan extended period of time. A biomaterial may also be an autograft,allograft, or xenograft which can be used as a transplant material. Allthese materials that have found applications in other medical orbiomedical fields can also be used in the present invention.

As used herein, the term “microelectronic technology or process”generally encompasses the technologies or processes used for fabricatingmicro-electronic and optical-electronic components. Examples includelithography, etching (e.g., wet etching, dry etching, or vapor etching),oxidation, diffusion, implantation, annealing, film deposition,cleaning, direct-writing, polishing, planarization (e.g., by chemicalmechanical polishing), epitaxial growth, metallization, processintegration, simulation, or any combinations thereof. Additionaldescriptions on microelectronic technologies or processes can be foundin, e.g., Jaeger, Introduction to Microelectronic Fabrication, 2^(nd)Ed., Prentice Hall, 2002; Ralph E. Williams, Modern GaAs ProcessingMethods, 2^(nd) Ed., Artech House, 1990; Robert F. Pierret, AdvancedSemiconductor Fundamentals, 2^(nd) Ed., Prentice Hall, 2002; S.Campbell, The Science and Engineering of Microelectronic Fabrication,2^(nd) Ed., Oxford University Press, 2001, the contents of all of whichare incorporated herein by reference in their entireties.

As used herein, the term “selective” as included in, e.g., “patterningmaterial B using a microelectronics process selective to material A”,means that the microelectronics process is effective on material B butnot on material A, or is substantially more effective on material B thanon material B (e.g., resulting in a much higher removal rate on materialB than on material A and thus removing much more material B thanmaterial A).

As used herein, the term “carbon nano-tube” generally refers to asallotropes of carbon with a cylindrical nanostructure. See, e.g., CarbonNanotube Science, by P. J. F. Harris, Cambridge University Press, 2009,for more details about carbon nano-tubes.

Through the use of a single micro-device or a combination ofmicro-devices integrated into a disease detection apparatus, the diseasedetection capabilities can be significantly improved in terms ofsensitivity, specificity, speed, cost, apparatus size, functionality,and ease of use, along with reduced invasiveness and side-effects. Alarge number of micro-device types capable of measuring a wide range ofmicroscopic properties of biological sample for disease detection can beintegrated and fabricated into a single detection apparatus usingmicro-fabrication technologies and novel process flows disclosed herein.While for the purposes of demonstration and illustration, a few novel,detailed examples have been shown herein on how microelectronics ornano-fabrication techniques and associated process flows can be utilizedto fabricate highly sensitive, multi-functional, and miniaturizeddetection devices, the principle and general approaches of employingmicroelectronics and nano-fabrication technologies in the design andfabrication of high performance detection devices have been contemplatedand taught, which can and should be expanded to various combination offabrication processes including but not limited to thin film deposition,patterning (lithography and etch), planarization (including chemicalmechanical polishing), ion implantation, diffusion, cleaning, variousmaterials, and various process sequences and flows and combinationsthereof.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 (a) is a perspective illustration of a disease detectionapparatus of this invention in which a biological sample placed in it ormoving through it can be tested. FIG. 1(b) and FIG. 1(c) illustrate theapparatus which comprises multiple individual detection micro-devices.

FIG. 2 (a) is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention with multiple micro-devices. Abiological sample is placed in the apparatus or moving through it whileone or more microscopic properties of this biological sample aremeasured with the multiple micro-devices. FIGS. 2(b)-2(l) areperspective illustration of the novel process flow for fabricating themicro-device. FIGS. 2(m)-2(n) are cross-sectional views of an apparatuscomprising multiple individual micro-devices.

FIG. 3 is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention with multiple micro-devices ofdifferent detection probes. A biological sample is placed in theapparatus or moving through it and one or more microscopic properties ofthis sample are measured with the multiple micro-device.

FIG. 4 is a perspective illustration of a disease detection apparatus ofthis invention. It includes two slabs separated by a narrow spacing witha biological sample to be analyzed placed between the slabs, withmultiple micro-devices placed at the inner surfaces of the slabs tomeasure one or more desired parameters of the sample at microscopiclevels.

FIG. 5 illustrates a novel process flow for fabricating a diseasedetection apparatus of this invention utilizing microelectronicstechnologies.

FIG. 6 is a perspective illustration of a disease detection apparatusfabricated by a method of this invention. The apparatus is capable ofprobing a single cell and measuring its microscopic properties.

FIG. 7 is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention with multiple micro-devices placedat a desired distance for time of flight measurements with enhancedsensitivity, specificity, and speed, including time dependent or dynamicinformation.

FIG. 8 is a perspective illustration of a novel set of microscopicprobes, included in a disease detection apparatus of this invention, fordetecting various electronic or magnetic states, configurations, orother properties of a biological sample (e.g., a cell, a DNA or RNAmolecule, a telomere of DNA or chromosome, a virus, or a tissue sample).

FIG. 9 is a perspective illustration of a novel four-point probe,included in a disease detection apparatus of this invention, fordetecting weak electronic signal in a biological sample (e.g., a cell, aDNA or RNA molecule, a telomere of DNA or chromosome, a virus, or atissue sample).

FIG. 10 illustrates a novel process flow for fabricating a class ofmicro-devices capable of trapping, sorting, probing, measuring, andmodifying a biological subject (e.g., a cell, a DNA or RNA molecule, atelomere of DNA or chromosome, a virus, or a tissue sample) at themicroscopic level and in a three-dimensional space.

FIG. 11 illustrates a novel process flow for fabricating a class ofmicro-devices capable of measuring physical properties of a biologicalsubject (e.g., a cell, a DNA or RNA molecule, a telomere of DNA orchromosome, a virus, or a tissue sample) such as mechanical properties(e.g., hardness, shear strength, elongation strength, fracture stress)and other properties related to cell membrane.

FIG. 12 illustrates how a micro-device with two micro-probes capable ofmoving in opposite directions when a force is applied can be utilized toprobe properties of a biological subject (e.g., mechanical properties ofa cell membrane).

FIG. 13 illustrates a novel time of flight detection arrangement fordisease detection applications, in which both clock signal generator andsignal detection probes are used, along with schematically recordedclock signal, probe signal (signal detected by probing micro-device),and processed and enhanced signal after signal filtering using phaselock-in processing technique to enhance the detected signal.

FIG. 14 illustrates yet another time of flight disease detectionarrangement in which clock signal generators, a probe signal generator,and signal detection probes are used, along with schematically recordedclock signal, detected signal by probing micro-device in response toprobe signal, and processed and enhanced signal after signal filteringusing phase lock-in processing technique to enhance the detected signalshowing detected response signal as a function of time (response signaldelays over time in this case).

FIG. 15 illustrates another novel time of flight disease detectionapplication, in which a set of novel micro-filters are utilized todetect biological subjects via separation of biological subjects bytheir various, specific properties such as size, weight, shape,electrical properties, or surface properties.

FIG. 16 illustrates a fluid delivery system, which is a pretreatmentpart for the disease detection apparatus, and it delivers a sample orauxiliary material at a desired pressure and speed into a device.

FIGS. 17(b)-17(c) illustrate a novel device which can engage in cellularcommunications at the single cell level by simulating cellular signalsand receiving the cell's responses which can be a signal of electric,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property. FIG.17(a) illustrates how the signal is processed and responded in a singlecell.

FIG. 18 illustrates a system block diagram of a disease detectionapparatus, comprising various functional modules.

FIG. 19 illustrates a micro-device capable of communicating, trapping,sorting, analyzing, treating, or modifying a DNA and measuring the DNA'svarious properties (e.g., electric, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical properties).

FIG. 20 illustrates an apparatus of this invention that can detect thesurface charge on biological subjects and separate them by a slit basedon the charge.

FIG. 21 illustrates another apparatus of this invention that can detectthe optical properties of the biological subject with a set of opticalsensors.

FIG. 22 illustrates another apparatus of this invention that canseparate biological subjects of different geometric size and detecttheir properties respectively.

FIG. 23 illustrates an apparatus of this invention that can measure theacoustic property of a biological subject.

FIG. 24 illustrates an apparatus of this invention that can measure theinternal pressure of a biological subject.

FIG. 25 illustrates an apparatus of this invention that has concavesbetween the probe couples, in the bottom or ceiling of the channel.

FIG. 26 illustrates another apparatus of this invention that hasconcaves of a different shape from those illustrated in FIG. 25.

FIG. 27 illustrates an apparatus of this invention that has a steppedchannel.

FIG. 28 illustrates an apparatus of this invention that has a set ofthermal meters.

FIG. 29 illustrates an apparatus of this invention that includes acarbon nano-tube as the channel with DNA contained therein.

FIG. 30 illustrated an integrated apparatus of this invention thatincludes a detecting device and an optical sensor.

FIG. 31 illustrated an integrated apparatus of this invention thatincludes a detecting device and a logic circuitry.

FIG. 32 illustrates an apparatus of this invention that includes adetecting device and a filter.

FIG. 33 illustrates how micro-devices of this invention can be used tomeasure the geometric factors of DNA.

FIG. 34 illustrates a process for fabricating a micro-device of thisinvention with a cover atop the trench to form a channel.

FIG. 35 is a diagram of an apparatus of this invention for detecting adisease in a biological subject.

FIG. 36 shows an example of a sample filtration unit.

FIG. 37 shows another example of a sample filtration unit.

FIG. 38 is a diagram of a pre-processing unit of an apparatus of thisinvention.

FIG. 39 is a diagram of an information processing unit of an apparatusof this invention.

FIG. 40 shows the integration of multiple signals which results incancellation of noise and enhancement of signal/noise ratio.

FIG. 41 shows one embodiment of the fabrication process of thisinvention for manufacturing a detection device with at least onedetection chamber and at least one detector.

FIG. 42 shows another embodiment of a process of this invention formanufacturing a detection device with enclosed detection chambers,detectors, and channels for transporting biological samples such asfluidic samples.

FIG. 43 shows a novel disease detection method in which at least oneprobe object is launched at a desired speed and direction toward abiological subject, resulting in a collision.

FIG. 44 illustrates a novel fabrication process of this invention forforming multiple components with different materials at the same devicelevel.

FIG. 45 shows a process of this invention for detecting a biologicalsubject using a disease detection device.

FIG. 46 shows another embodiment of disease detection process whereindiseased and healthy biological subjects are separated and the diseasedbiological subjects are delivered to further test.

FIG. 47 is an arrayed biological detecting device wherein a series ofdetecting devices are fabricated into an apparatus.

FIG. 48 shows another embodiment of a disease detection device of thecurrent invention including inlet and outlet of the device, the channelwhere the biological subject passes through, and detection devicesaligned along the walls of the channel.

FIG. 49 shows a schedule for fabricating a piezo-electric micro-detectorof this invention.

FIG. 50 shows an example of the micro-device of this invention packagedand ready for use.

FIG. 51 shows another example of the micro-device of this invention thatis packaged and ready for use.

FIG. 52 shows yet another example of the micro-device of this inventionthat is packaged and ready for use.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to apparatus for detectingdisease in a biological subject in vivo or in vitro (e.g., human being,an organ, a tissue, or cells in a culture). Each apparatus includes abiological fluid delivering system and a probing and detecting device.The apparatus is capable of measuring microscopic properties of abiological sample. By the constant pressure fluid delivery system,microscopic biological subjects can be delivered onto or into thediagnostic micro-device of the apparatus. Compared to traditionaldetection apparatus or technologies, the apparatus provided by thisinvention are advantageous in providing enhanced detection sensitivity,specificity, and speed, with reduced costs and size. The apparatus canfurther include a biological interface, a probing controlling and dataanalysis circuitry, or a system reclaiming or treating medical waste.Additional micro-devices, e.g., a second detection device, can also beincluded or integrated into the apparatus for enhanced detectioncapabilities.

As a key component of the apparatus, the micro-device should includemeans to perform at least the function of addressing, controlling,forcing, receiving, amplifying, or storing information from each probingaddress. As an example, such means can be a central control unit thatincludes a controlling circuitry, an addressing unit, an amplifiercircuitry, a logic processing circuitry, a memory unit, an applicationspecific chip, a signal transmitter, a signal receiver, or a sensor.

In some embodiments, the fluid delivering system comprises a pressuregenerator, a pressure regulator, a throttle valve, a pressure gauge, anddistributing kits. As examples of these embodiments, the pressuregenerator can include a motor piston system and a bin containingcompressed gas; the pressure regulator (which can consist of multipleregulators) can down-regulate or up-regulate the pressure to a desiredvalue; the pressure gauge feeds back the measured value to the throttlevalve which then regulates the pressure to approach the target value.

The biological fluid to be delivered can be a sample of a biologicalsubject to be detected for disease or something not necessarily to bedetected for disease. In some embodiment,; the fluid to be delivered isliquid (e.g., a blood sample, a urine sample, or a saline) or gas (e.g.,nitrogen, argon, helium, neon, krypton, xenon, or radon). The pressureregulator can be a single pressure regulator or multiple pressureregulators which are placed in succession to either down-regulate orup-regulate the pressure to a desired level, particularly when theinitial pressure is either too high or too low for a single regulator toadjust to the desired level or a level that is acceptable for an enddevice or target.

In some other embodiments, the system controller includes apre-amplifier, a lock-in amplifier, an electrical meter, a thermalmeter, a switching matrix, a system bus, a nonvolatile storage device, arandom access memory, a processor, or a user interface. The interfacecan include a sensor which can be a thermal sensor, a flow meter, apiezo-meter, or another sensor.

In still some other embodiments, apparatus of this invention furtherinclude a biological interface, a system controller, a system forreclaiming or treatment medical waste. The reclaiming and treatment ofmedical waste can be performed by the same system or two differentsystems.

Another aspect of this invention provides apparatus for interacting witha cell, which include a device for sending a signal to the cell andoptionally receiving a response to the signal from the cell.

In some embodiments, the interaction with the cell can be probing,detecting, communicating with, treating, or modifying with a codedsignal that can be an electric, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical signal, or acombination thereof.

In some other embodiments, the device contained in the apparatus caninclude multiple surfaces coated with one or more elements orcombinations of elements, and a control system for releasing theelements. In some instances, the control system can cause release of theelements from the device surface via thermal energy, optical energy,acoustic energy, electrical energy, electro-magnetic energy, magneticenergy, radiation energy, or mechanical energy in a controlled manner.The energy can be in the pulsed form at desired frequencies.

In some other embodiments, the device contained in the apparatus includea first component for storing or releasing one element or a combinationof elements onto the surface of the cell or into the cell; and a secondcomponent for controlling the release of the elements (e.g., a circuitryfor controlling the release of the elements). The elements can be abiological component, a chemical compound, Ca, C, Cl, Co, Cu, H, I, Fe,Mg, Mn, N, O, P, F, K, Na, S, Zn, or a combination thereof. The signal,pulsed or constant, can be in the form of a released element orcombination of elements, and it can be carried in a liquid solution,gas, or a combination thereof. In some instances, the signal can be at afrequency ranging from about 1×10⁻⁴ Hz to about 100 MHz or ranging fromabout 1×10⁻⁴ Hz to about 10 Hz, or at an oscillation concentrationranging from about 1.0 nmol/L to about 10.0 mmol/L. Also, the signalcomprises the oscillation of a biological component, a chemicalcompound, Ca, C, Cl, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na, S, Zn,or a combination thereof, e.g., at desired oscillating frequencies.

In some embodiments, the signal to be sent to the cell can be in theform of oscillating element, compound, or an oscillating density of abiological component, and a response to the signal from the cell is inthe form of oscillating element, compound, or an oscillating density ofa biological component.

In some embodiments, the device can be coated with a biological film,e.g., to enhance compatibility between the device and the cell.

In some other embodiments, the device can include components forgenerating a signal to be sent to the cell, receiving a response to thesignal from the cell, analyzing the response, processing the response,and interfacing between the device and the cell.

Still another aspect of this invention provides devices each including amicro-filter, a shutter, a cell counter, a selector, a micro-surgicalkit, a timer, and a data processing circuitry. The micro-filter candiscriminate abnormal cells by a physical property (e.g., e.g.,dimension, shape, or velocity), mechanical property, electric property,magnetic property, electromagnetic, thermal property (e.g.,temperature), optical property, acoustical property, biologicalproperty, chemical property, or bio-chemical property. The devices eachcan also include one or more micro-filters. Each of these micro-filterscan be integrated with two cell counters, one of which is installed atthe entrance of each filter well, while the other is installed at theexit of each filter well. The shape of the micro-filter's well isrectangle, ellipse, circle, or polygon; and the micro-filter's dimensionranges from about 0.1 μm to about 500 μm or from about 5 um to about 200um. As used herein, the term “dimension” means the physical or featuresize of the filter opening, e.g., diameter, length, width, or height.The filter can be coated with a biological or bio-compatible film, e.g.,to enhance compatibility between the device and the cell.

In some embodiments of these devices, the shutter sandwiched by twofilter membranes can be controlled by a timer (thus time shutter). Thetimer can be triggered by the cell counter. For instance, when a cellpasses through the cell counter of the filter entrance, the clock istriggered to reset the shutter to default position, and moves at apreset speed towards the cell pathway, and the timer records the time asthe cell pass through the cell counter at the exit.

Still a further aspect of this invention provides methods forfabricating a micro-device with micro-trench and probe embedded in themicro-trench's sidewalls. A micro-trench is an unclosed tunnel (see,e.g., FIG. 2(i), 2030), which can be coupled with another upendedsymmetric trench (see, e.g., FIG. 2(k), 2031) to form a closed channel(see, e.g., FIG. 2(l), 2020). The method may include chemical vapordeposition, physical vapor deposition, or atomic layer deposition todeposit various materials on a substrate; lithography or etch totransfer patterns from design to structure; chemical mechanicalplanarization for surface planarization, chemical cleaning for particleremoval, diffusion or ion implantation for doping elements into specificlayers; or thermal anneal to reduce the crystal defects and activatediffused ions. An example of such method includes: depositing a firstmaterial onto a substrate; depositing a second material onto the firstmaterial and patterning the second material by a microelectronic process(e.g., lithography or etch) to form a detecting tip; depositing a thirdmaterial on the second material and then patterning the second materialby a planarization process; depositing a fourth material on the thirdmaterial and patterning the fourth material first by a microelectronicprocess (e.g., lithography or etch) and then by a microelectronicprocess (e.g., another etch) in which the fourth material serves as ahardmask. A hardmask generally refers to a material (e.g., inorganicdielectric or metallic compound) used in semiconductor processing as anetch mask in lieu of polymer or other organic “soft” materials.

In some embodiments, the method further includes coupling two devicesthat are thus fabricated and symmetric (i.e., a flipped mirror) to forma detecting device with channels. The entrance of each channel can beoptionally bell-mouthed, e.g., such that the size of channel's openingend (the entrance) is larger than the channel's body, thereby making iteasier for a cell to enter the channel. The shape of each channel'scross-section can be rectangle, ellipse, circle, or polygon. Themicro-trenches of the coupled two micro-devices can be aligned by themodule of alignment marks designed on the layout of the micro-device.The dimension of the micro-trench can range from about 0.1 um to about500 um.

Alternatively, the method can also include covering the micro-trench ofthe micro-device with a flat panel. Such a panel can comprise or be madewith silicon, SiGe, SiO₂, Al₂O₃, or other optical materials. Examples ofother potentially suitable optical materials include acrylate polymer,AgInSbTe, synthetic alexandrite, arsenic triselenide, arsenictrisulfide, barium fluoride, CR-39, cadmium selenide, caesium cadmiumchloride, calcite, calcium fluoride, chalcogenide glass, galliumphosphide, GeSbTe, germanium, germanium dioxide, glass code, hydrogensilsesquioxane, Iceland spar, liquid crystal, lithium fluoride,lumicera, METATOY, magnesium fluoride, agnesium oxide, negative indexmetamaterials, neutron super mirror, phosphor, picarin, poly(methylmethacrylate), polycarbonate, potassium bromide, sapphire, scotophor,spectralon, speculum metal, split-ring resonator, strontium fluoride,yttrium aluminum garnet, yttrium lithium fluoride, yttriumorthovanadate, ZBLAN, zinc selenide, and zinc sulfide.

In other embodiments, the method can further include integrating threeor more micro-devices thus fabricated to yield an enhanced device withan array of the channels.

Yet still another aspect of this invention relates to micro-devices eachincluding a micro-trench, a probe embedded aside the trench's side wallsor bottom floor, a supporting structure to move the probe, and acontrolling circuitry, wherein the micro-device is capable of trapping,sorting, or modifying a DNA and measuring its properties (e.g.,electrical, thermal, or optical properties). The micro-trench can beutilized to encase the DNA double helix.

In some embodiments, the width of the micro-trench ranges from about 1nm to about 10 μm, the depth of the micro-trench ranges from about 1 nmto about 10 μm, or the length of the micro-trench ranges from about 1 nmto about 10 mm. The probe can include or be made of a conductivematerial and, optionally, a flexible supporting structure to extend orcontract the probe. The probe can also have a tip aside the trench andthe tip matches spatially with either a major groove or a minor grooveof the DNA. The tip can match spatially with interlaced grooves of theDNA, which can be variable. The tip of can also match the end of eachstrand of the DNA helix. In some examples, the tip's diameter can rangefrom about 1 angstrom to about 10 μm.

In some other embodiments, the micro-device can further include an arrayof trenches, e.g., to enhance the efficiency.

Another aspect of this invention relates to a set of novel process flowsfor fabricating micro-devices (including micro-probes andmicro-indentation probes) for their applications in disease detection bymeasuring microscopic properties of a biological sample. Themicro-devices can be integrated into a disease detection apparatus ofthis invention to measure one or more properties at microscopic levels.

Another aspect of this invention is to involve in cellularcommunications and regulate cellular decision or response (such asdifferentiation, dedifferentiation, cell division and cell death) withfabricated signals. This could be further employed to detect and treatdiseases.

To further enhance measurement capabilities, multiple micro-devices canbe implemented into a piece of detection apparatus employing the time offlight technique, in which at least one probing micro-device and onesensing micro-device placed at a preset, known distance. The probingmicro-device can apply a signal (e.g., a voltage, a charge, anelectrical field, a laser beam, or an acoustic wave) to the biologicalsample to be measured, and the detection (sensing) micro-device canmeasure response from or of the biological sample after the sample hastraveled a known distance and a desired period of time. For instance, aprobing micro-device can apply an electrical charge to a cell first, andthen a detection (sensing) micro-device subsequently measures thesurface charge after a desired period of time (T) has lapsed and thecell has traveled a certain distance (L).

The micro-devices contained in the apparatus of this invention can havea wide range of designs, structures, functionalities, and applicationsdue to their diverse properties, high degree of flexibilities, andability of integration and miniaturization. They include, e.g., avoltage comparator, a four point probe, a calculator, a logic circuitry,a memory unit, a micro cutter, a micro hammer, a micro shield, a microdye, a micro pin, a micro knife, a micro needle, a micro thread holder,micro tweezers, a micro optical absorber, a micro mirror, a microwheeler, a micro filter, a micro chopper, a micro shredder, micro pumps,a micro absorber, a micro signal detector, a micro driller, a microsucker, a micro tester, a micro container, a signal transmitter, asignal generator, a friction sensor, an electrical charge sensor, atemperature sensor, a hardness detector, an acoustic wave generator, anoptical wave generator, a heat generator, a micro refrigerator and acharge generator.

Further, it should be noted that advancements in manufacturingtechnologies have now made fabrications of a wide range of micro-devicesand integration of various functions onto the same device highlyfeasible and cost effective. The typical human cell size is about 10microns. Using state-of-the-art integrated circuit fabricationtechniques, the minimum feature size defined on a micro-device can be assmall as 0.1 micron or below. Thus, it is ideal to utilize the disclosedmicro-devices for biological applications.

In terms of materials for the micro-devices, the general principle orconsideration is the material's compatibility with a biological subject.Since the time in which a micro-device is in contact with a biologicalsample (e.g., a cell; a biological molecule such as DNA, RNA, orprotein; or a tissue or organ sample) may vary, depending on itsintended application, a different material or a different combination ofmaterials may be used to make the micro-device. In some special cases,the materials may dissolve in a given pH in a controlled manner and thusmay be selected as an appropriate material. Other considerations includecost, simplicity, ease of use and practicality. With the significantadvancements in micro fabrication technologies such as integratedcircuit manufacturing technology, highly integrated devices with minimumfeature size as small as 0.1 micron can now be made cost-effectively andcommercially. One good example is the design and fabrication of microelectro mechanical devices (MEMS), which now are being used in a widevariety of applications in the integrated circuit industry.

Set forth below are several illustrations or examples of apparatus ofthis invention containing a class of innovative micro-devices that areintegrated into the disease detection apparatus of this invention, andof their fabrication process.

FIG. 1 is a perspective illustration of a disease detection apparatus ofthis invention 111 in which a biological sample 211 such as a bloodsample placed in it or moving through it is tested. In this figure, anexample of disease detection apparatus 111 is in the form of a cylinder,in which a biological sample 211 flowing through it (from the left sideto the right side in the figure) can be tested for one or moreproperties at the microscopic levels.

To enhance detection speed and sensitivity, a large number ofmicro-devices can be integrated into a single disease detectionapparatus of this invention, such as the apparatus illustrated in FIG.1(b) and FIG. 1(c) with the micro-devices spaced to measure a largenumber of desired entities (such as cells, DNAs, RNAs, proteins, etc.)in the biological sample. To achieve the above requirements, thedetection apparatus should be optimized with its surface area maximizedto contact the biological sample and with large number of micro-devicesintegrated on the maximized surface.

FIG. 2 (a) is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention 122 with multiple identicalmicro-devices 311. A biological sample such as a blood sample 211 placedin it or moving through it can be tested for one or more properties atthe microscopic levels including, e.g., electrical properties (such assurface charge, surface potential, current, impedance, other electricalproperties), magnetic properties, electromagnetic properties, mechanicalproperties (such as density, hardness, shear strength, elongationstrength, fracture tress, and adhesion), biological features, chemicalproperties (e.g., pH or ionic strength), biochemical properties, thermalproperties (e.g., temperature), and optical properties.

Instead of measuring a single property of a biological subject fordisease diagnosis, various micro-devices can be integrated into adetection apparatus to detect multiple properties. FIG. 3 is aperspective, cross-sectional illustration of a disease detectionapparatus of this invention 133 with multiple micro-devices 311, 312,313, 314, and 315, of different detection probes in which a sample 211such as a blood sample placed in it or moving through it can be testedfor multiple properties including but not limited to electricalproperties (e.g., surface charge, surface potential, and impedance),magnetic properties, electromagnetic properties, mechanical properties(e.g., density, hardness and adhesion), thermal properties (e.g.,temperature), biological properties, chemical properties (e.g., pH),physical properties, acoustical properties, and optical properties.

FIGS. 2(b)-2(n) illustrate a process flow of this invention forfabricating micro-devices for trapping, sorting, probing, measuring, andmodifying biological subjects (e.g., a single cell, a DNA or RNAmolecule). First, a material 2002 (e.g., a non-conducting material) andanother material 2003 (e.g., a conducting material) are sequentiallydeposited on a substrate 2001 (see FIG. 2(b) and FIG. 2(c)). The firstmaterial 2003 is then subsequently patterned by the lithography and etchprocesses (see FIG. 2(d)). Another material 2004 is then deposited (asshown in FIG. 2(e)) and planarized (as shown in FIG. 2(f)). Anotherlayer of material 2005 is deposited (as shown in FIG. 2(g)) andpatterned as a hard mask (as shown in FIG. 2(h)), then followed by etch(as shown in FIG. 2(j)), which is stopped on the substrate 2001. FIG.2(i) is a perspective illustration of the device, while FIG. 2(j) is avertical illustration of the device.

As shown in FIG. 2(k), the device 2080 and a mirrored or symmetricdevice 2081 can be coupled together (as shown in FIG. 2(l)). As such,the apparatus having the pathway with probe embedded in the sidewall isfabricated.

As illustrated in FIG. 2(m) and FIG. 2(n), a large number of detectionmicro-devices can be integrated together to enhance the detectionefficiency.

As illustrated herein, it is desirable to optimize the detectionapparatus design to maximize measurement surface area, since the greaterthe surface area, the greater number of micro-devices that can be placedon the detection apparatus to simultaneously measure the sample, therebyincreasing detection speed and also minimizing the amount of sampleneeded for the test. FIG. 4 is a perspective illustration of a diseasedetection apparatus of this invention 144. It includes two slabsseparated by a narrow spacing with a sample such as a blood sample to bemeasured placed between the slabs, with multiple micro-devices placed atthe inner surfaces of the slabs to measure one or more properties of thesample at microscopic levels.

Yet another aspect of this invention relates to a set of novelfabrication process flows for making micro-devices for disease detectionpurposes. FIG. 5 illustrates a novel process flow for fabricating adisease detection apparatus utilizing microelectronics technologies andprocesses. First, a material 412 is deposited on a substrate 411 (FIG.5(a)). It is then patterned by photolithography and etching processes(FIG. 5(b)). Following the deposition, material 413 is planarized usingchemical mechanical polishing as shown in FIG. 5(d). Recessed areas, inthe form of hole pattern, are next formed in material 413 usingphotolithography and etch processes, as shown in FIG. 5(e), followed bythe deposition of material 414 (FIG. 5(f)). Material 414 above thesurface of material 413 is removed by chemical mechanical polishing(FIG. 5(g), followed by deposition of material 415. Material 415 is nextpatterned using photolithography and etching processes (FIG. 5(i)).Material 414 is next deposited and its excess material above itssubstrate 415 is removed by chemical mechanical polishing (FIG. 5(j) and(k)). Finally, a light etch or short chemical mechanical polishing tomaterial 415 is carried out to recess material 415, selective tomaterial 414 (FIG. 5(l)), resulting in slight protruding of material414. Material 412 can be a piezo-electric material. When a voltage isapplied to it in the right direction, it will expand and push up,resulting in upward motion in middle tip in material 414. Thus, amicro-device with two probes capable of measuring a range of properties(including mechanical and electrical properties) of biological samplesis fabricated, using the above novel fabrication process flow.

Detection apparatus integrated with micro-devices disclosed in thisapplication is fully capable of detecting pre-chosen properties on asingle cell, a single DNA, a single RNA, or an individual, small sizedbiological matter level. FIG. 6 is a perspective illustration of amicro-device 555 fabricated by a novel process flow disclosed in thispatent application (e.g., novel process flow illustrated in FIG. 5above) and how such a device is capable of probing a single cell 666 andmeasuring the cell for collecting intended parameters. FIG. 6(a)illustrated a perspective, cross-section of a micro-device 555 with apair of micro probes 531 and 520, where micro probe 531 is in the formof a tip and micro probe 520 is in the form of a ring. Both of microprobes 531 and 520 can be conductive and they can serve as a pair ofprobes to measure electrical properties of a biological sample. Microprobe 531 is in contact with a base 518 which can be a piezo-electricmaterial. When a voltage is applied to the base 518 made of apiezo-electric material, the base 518 can expand and push micro probetip 531 upward, which can be useful in measuring various properties of abiological sample such as a single cell. In FIG. 6(b), micro-device 555is shown to measure a single cell 666, using probe tip 531 penetratingthrough cell membrane 611 and into the cell's inner space 622, whileprobe ring 520 making contact with cell membrane 611 at the outsidesurface of the membrane. This way, the micro-device 555 can make variousmeasurements on the cell, including its electrical properties (e.g.,electrical potential, current across the cell membrane, surface chargeon the membrane, and impedance), mechanical properties (e.g., hardnesswhen probe tip 531 is designed as a micro-indentation probe), thermalproperties (e.g., temperature), physical properties, and chemicalproperties (e.g., pH).

In another further aspect, the invention provides the design,integration, and fabrication process flow of micro-devices capable ofmaking highly sensitive and advanced measurements on very weak signalsin biological systems for disease detection under complicatedenvironment with very weak signal and relatively high noise background.Those novel capabilities using the class of micro-devices disclosed inthis invention for disease detection include but not limited to makingdynamic measurements, real time measurements (such as time of flightmeasurements, and combination of using probe signal and detectingresponse signal), phase lock-in technique to reduce background noise,and 4-point probe techniques to measure very weak signals, and uniqueand novel probes to measure various electronic, electromagnetic andmagnetic properties of biological samples at the single cell (e.g., atelomere of DNA or chromosome), single molecule (e.g., DNA, RNA, orprotein), single biological subject (e.g., virus) level.

For example, in a time of flight approach to obtain dynamic informationon the biological sample (e.g., a cell, a substructure of a cell, a DNA,a RNA, or a virus), a first micro-device is first used to send a signalto perturb the biological subject to be diagnosed, and then a secondmicro-device is employed to accurately measure the response from thebiological subject. In one embodiment, the first micro-device and thesecond micro-device are positioned with a desired or pre-determineddistance L apart, with a biological subject to be measured flowing fromthe first micro-device towards the second micro-device. When thebiological subject passes the first micro-device, the first micro-devicesends a signal to the passing biological subject, and then the secondmicro-device detects the response or retention of the perturbationsignal on the biological subject. From the distance between the twomicro-devices, time interval, the nature of perturbation by the firstmicro-device, and measured changes on the biological subject during thetime of flight, microscopic and dynamic properties of the biologicalsubject can be obtained. In another embodiment, a first micro-device isused to probe the biological subject by applying a signal (e.g., anelectronic charge) and the response from the biological subject isdetected by a second micro-device as a function of time.

To further increase detection sensitivity, a novel detection process fordisease detection is used, in which time of flight technique isemployed. FIG. 7 is a perspective, cross-sectional illustration ofdetection apparatus 155 with multiple micro-devices 321 and 331 placedat a desired distance 700 for time of flight measurements to attaindynamic information on biological sample 211 (e.g., a cell) withenhanced measurement sensitivity, specificity, and speed. In this timeof flight measurement, one or more properties of the biological sample211 are first measured when the sample 211 passes the first micro-device321. The same properties are then measured again when the sample 211passes the second micro-device 331 after it has travelled the distance700. The change in properties of sample 211 from at micro-device 321 toat micro-device 331 indicates how it reacts with its surroundingenvironment (e.g., a particular biological environment) during thatperiod. It may also reveal information and provide insight on how itsproperties evolve with time. Alternatively, in the arrangement shown inFIG. 7, micro-device 321 could be used first as a probe to apply a probesignal (e.g., an electrical charge) to sample 211 as the sample passesthe micro-device 321. Subsequently, the response of the sample to theprobe signal can be detected by micro-device 331 as the sample passes it(e.g., change in the electrical charge on the sample during the flight).Measurements on biological sample 211 can be done via contact ornon-contact measurements. In one embodiment, an array of micro-devicescan be deployed at a desired spacing to measure properties of thebiological subject over time.

The utilization of micro-devices (e.g., made by using the fabricationprocess flows of this invention) as discussed above and illustrated inFIG. 7 can be helpful for detecting a set of new, microscopic propertiesof a biological sample (e.g., a cell, a cell substructure, or abiological molecule such as DNA or RNA or protein) that have not beenconsidered in existing detection technologies. Such microscopicproperties can be electric, magnetic, electromagnetic, thermal, optical,acoustical, biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical properties of abiological sample that is a single biological subject (such as a cell, acell substructure, a biological molecule—e.g., DNA, RNA, or protein—or asample of a tissue or organ). It is known that biological mattersincludes from basic bonding such as OH, CO, and CH bonding, to complex,three dimensional structures such as DNA and RNA. Some of them have aunique signature in terms of its electronic configuration. Some of themmay have unique electric, magnetic, electromagnetic, thermal, optical,acoustical, biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical properties andconfigurations. Normal biological subject and diseased biologicalsubject may carry different signatures with respective to the above saidproperties. However, none of the above stated parameters or propertieshave been routinely used as a disease detection property. Using adisease detection apparatus including one or more micro-devices of thisinvention, those properties can be detected, measured, and utilized asuseful signals for disease detection, particularly for early stagedetection of serious diseases such as cancer.

FIG. 8 is a perspective illustration of a novel set of microscopicprobes 341, 342, 343, 344, 345, 346, and 347 designed and configured todetect various electronic, magnetic, or electromagnetic states,configurations, or other properties at microscopic level on biologicalsamples 212, 213, 214, and 215, which can be a single cell, DNA, RNA,and tissue or sample. As an example, in terms of measuring electronicproperties, the shapes of biological samples 212, 213, 214, and 215 inFIG. 8 may represent electronic monopole (sample 212), dipole (samples213 and 214), and quadruple (sample 215). The micro-devices 341, 342,343, 344, 345, 346, and 347 are optimized to maximize measurementsensitivity of those said parameters including but not limited toelectronic states, electronic charge, electronic cloud distribution,electrical field, and magnetic and electromagnetic properties, and themicro-devices can be designed and arranged in three dimensionalconfigurations. For some diseases such as cancer, it is likely thatelectronic states and corresponding electronic properties differ betweennormal and cancerous cells, DNA, RNA, and tissue. Therefore, bymeasuring electronic, magnetic and electromagnetic properties atmicroscopic levels including at cell, DNA, and RNA levels, diseasedetection sensitivity and specificity can be improved.

In addition to the above examples in measuring electrical properties(e.g., charge, electronic states, electronic charge, electronic clouddistribution, electrical field, current, and electrical potential, andimpedance), mechanical properties (e.g., hardness, density, shearstrength, and fracture strength) and chemical properties (e.g., pH) in asingle cell, and in FIG. 8 for measuring electrical, magnetic orelectromagnetic states or configurations of biological samples at celland biological molecular (e.g., DNA, RNA, and protein) levels, othermicro-devices are disclosed in this application for sensitive electricalmeasurements.

FIG. 9 is a perspective illustration of a four-point probe for detectingweak electronic signal in a biological sample such as a cell, where afour point probe 348 is designed to measure electrical properties(impedance and weak electrical current) of a biological sample 216.

One of the key aspects of this invention is the design and fabricationprocess flows of micro-devices and methods of use the micro-devices forcatching and/or measuring biological subjects (e.g., cells, cellsubstructures, DNA, and RNA) at microscopic levels and in threedimensional space, in which the micro-devices have micro-probes arrangedin three dimensional manner with feature sizes as small as a cell, DNA,or RNA, and capable of trapping, sorting, probing, measuring, andmodifying biological subjects. Such micro-devices can be fabricatedusing state-of-the-art microelectronics processing techniques such asthose used in fabricating integrated circuits. Using thin filmdeposition technologies such as molecular epitaxy beam (MEB) and atomiclayer deposition (ALD), film thickness as thin as a few monolayers canbe achieved (e.g., 4 A to 10 A). Further, using electron beam or x-raylithography, device feature size on the order of nanometers can beobtained, making micro-device capable of trapping, probing, measuring,and modifying a biological subject (e.g., a single cell, a single DNA orRNA molecule) possible.

FIG. 10 illustrates a process flow of this invention for fabricatingmicro-devices for trapping, sorting, probing, measuring, and modifyingbiological subjects (e.g., a single cell, a DNA or RNA molecule). Inthis process flow, microelectronics processes are utilized to fabricatemicro-devices designed to achieve the above stated unique functions.Specifically, a first material 712 (typically a conducting material) isfirst deposited on a substrate 711 (FIG. 10(a) and FIG. 10(b)). Thefirst material 712 is subsequently patterned by using lithography andetch processes (FIG. 10(c)). A second material 713 is then deposited andplanarized using chemical mechanical polishing process to removeoverburden of the second material 713 above the first material 712 (asshown in FIG. 10(e)). Another layer of material 714 is deposited andpatterned, followed by deposition and planarization by chemicalmechanical polishing of another layer of 712 (FIG. 10(f)). Next, a thirdmaterial 715 is deposited and patterned, using lithography and etchprocesses (FIG. 10(g) and FIG. 10(h)), followed by deposition andplanarization of a fourth material 716, typically a sacrificial material(FIG. 10(i) and FIG. 10(j)). Repeating the process flow of deposition ofpatterning material 712 or material 715 alternatively, and deposition ofmaterial 716 and planarization by chemical mechanical polishing (FIG.10(k)-(m)), a film stack featuring multiple layers with alternatingmaterial 712 (e.g., a conducting material) and material 715 (e.g., aninsulating material) in at least portions of the device is formed.Finally, material 716 between film stacks 771 and 772 is removed by wetetch, dry etch (which may require lithography process), or vapor etch,selective to all other materials (FIG. 10(n)). As illustrated in FIG.10(o), in the case of 712 being a conductive material connected to anelectrical circuit or an electrical source (e.g., a charge source), eachprobe tip formed by 712 on the stack (e.g., 781 and 782) can have acharge or an electrical field at the surface (e.g., 781 and 782), which(each probe tip) can be selected to have a positive charge or a negativecharge, or a positive electrical field or negative electrical field.Conversely, such probe tip can also sense various properties ofbiological subject being measured (e.g., electronic cloud, field,charge, or temperature when the probe tip is a thermal detector, orlight emission when the probe tip is an optical sensor). Usingelectrical circuit or electrical source, various combinations ofelectrical charge distribution or electrical field can be placed on themicro-device, as shown in FIG. 10(o) and FIG. 10(p), which can be usedto sort and trap various biological subjects such as a cell and a DNAmolecule. For instance, a biological subject with a charge distributioninverse of that in FIG. 10(p) can be trapped by the micro-device shownin FIG. 10(p). An array of micro-devices with various chargedistributions or electrical field distributions can trap theirrespective biological subjects in a high speed, which can serve as asorting device. FIG. 10(q) illustrates the use of a micro-device capableof trapping a DNA or measuring various properties (e.g., electrical,thermal, or optical properties) of a DNA, with each probe tip matched upspatially with either a major groove or minor groove of a double helixDNA. FIG. 10(r) illustrates how the probe tips are connected toelectrical circuit, where only electrical wiring is shown. It should benoted that the micro-device shown in this example can be integrated ontoa single chip with one billion or more such micro-devices to trap and/orsort cells, DNAs, RNAs, proteins, and other biological subject in a highspeed.

Another aspect of this invention relates to micro-indentation probes andmicro-probes for measuring a range of physical properties (such asmechanical properties) of biological subjects. Examples of themechanical properties include hardness, shear strength, elongationstrength, fracture stress, and other properties related to cell membranewhich is believed to be a critical component in disease diagnosis.

FIG. 11 illustrates a novel fabrication process flow for micro-devicescapable of probing a range of properties of biological subjects, such asmechanical properties of cell membrane (e.g., mechanical strength of acell membrane). In this process flow, a material 812 is first depositedonto a substrate 811, followed by the deposition of another material 813(FIG. 11(a)). Following patterning of material 813 using lithography andetch processes, a material 814 is deposited (FIG. 11(b)) and planarized(FIG. 11(c)). Another layer of material 813 is next deposited andpatterned using lithography and etch processes to remove portions of thematerial 813, followed by the deposition and planarization of a material815 (which can be a piezo-electric material and can serve as a driver)(FIG. 11(d)). A layer of material 813 is next deposited, followed bydeposition and patterning of yet another layer of 813, and depositionand planarization of material 816 (FIG. 11(e)). Next, material 816 isetched back to a reduced thickness, and patterned, followed bypatterning of triple-layer of material 813 (FIG. 11(f). Another layer of814 is deposited (FIG. 11(g)) and planarized by chemical mechanicalpolishing (FIG. 11(h)), and patterned (FIG. 11(i)). Finally, multiplelayers of 813 are removed by wet etch, plasma etch, or vapor etch (FIG.11(j)). FIG. 11(k) is a perspective, cross-sectional illustration of themicro-device in a plane perpendicular to that in FIG. 11(j) (90-degreerotation from FIG. 11(j)). FIG. 11(l) illustrates a micro-device withtwo micro-tips 871 and 872 which can move in opposite directions when avoltage is applied to piezo-electric drivers 881 and 882, which can beused to probe biological subjects such as cells.

FIG. 12 is an illustration of how micro-devices fabricated using thenovel manufacturing process shown in FIG. 11 work. In FIG. 12, amicro-device 850 with two micro-probes 866 and 855 can move in oppositedirections upon a force being applied (FIG. 12(a)). When the tips of thetwo probes are penetrated into a cell 870, as the distance between thetwo micro-probes is increased with the increasing applied force, thecell is stretched. Finally, as the applied force is reached a criticalvalue, the cell is broken into two pieces (FIG. 12(b)). The dynamicresponse of the cell to the applied force provides information on thecell, particularly on the mechanical properties (e.g., elasticity) ofcell membrane. The force at the point in which the cell is torn apartreflects the strength of the cell and it may be called a breaking point:the greater the mechanical strength of the cell membrane is, the greaterthe force is at the breaking point.

Another novel approach provided by this invention is the use of phaselock-in measurement for disease detection, which reduces backgroundnoise and effectively enhances signal to noise ratio. Generally, in thismeasurement approach, a periodic signal is used to probe the biologicalsample and response coherent to the frequency of this periodic probesignal is detected and amplified, while other signals not coherent tothe frequency of the probe signal is filtered out, which therebyeffectively reduces background noise. In one of the embodiments in thisinvention, a probing micro-device can send a periodic probe signal(e.g., a pulsed laser team, a pulsed thermal wave, or an alternatingelectrical field) to a biological subject, response to the probe signalby the biological subject can be detected by a detecting micro-device.The phase lock-in technique can be used to filter out unwanted noise andenhance the response signal which is synchronized to the frequency ofthe probe signal. The following two examples illustrate the novelfeatures of time of flight detection arrangement in combination withphase lock-in detection technique to enhance weak signal and thereforedetection sensitivity in disease detection measurements.

FIG. 13 is an illustration of a novel time of flight detectionarrangement for disease detection applications. Specifically, FIG. 13(a)shows a set-up for measuring biological subject 911 using detectionprobe 933 and clock generator 922, and FIG. 13(b) contains recordedsignal 921 due to structure 922, signal 931 recorded by signal probe933, and processed signal 941 using a phase lock-in technique to filterout noise in recorded signal 931, where only response synchronized toclock signal 921 is retained. In the setup shown in FIG. 13(a), when abiological subject such as a cell 911 passes a structure 922, ittriggers a clear signal (e.g., a light scattering signal if 922 is alight source, or a sharp increase in voltage if 922 is an orificestructure in a resistor). Therefore, 922 can be used to register thearrival of the biological subject, and as a clock when multiplestructures of 922 are placed at a periodic distance as shown in recordedsignal trace 921 in FIG. 13(b). In addition, when 922 is placed at aknown distance in front of a probe 933, it marks the arrival of abiological subject coming towards 933 and signal response recorded at933 is delayed by a time t from the signal triggered by 922 where tequals distance between 922 and 933 divided by traveling speed of thebiological subject. As illustrated in FIG. 13(b), signal 921 due tostructure 922 is clear and periodic with periodicity proportional todistance between structure 922 s, while signal measured by probe 933 hasa high noise level and relatively weak signal related to the biologicalsubject. With the utilization of phase lock-in technique to filter outnoise in recorded signal 931 by the detection probe 933 un-synchronizedto clock signal 921, signal to noise ratio can be greatly enhanced asshown in processed signal 941 in FIG. 13(b).

FIG. 14 illustrates yet another time of flight disease detectionarrangement in which a clock signal generator 922, a probe signalgenerator 944, and a signal detection probe 955 are used, along withschematically recorded clock signal 921, total recorded response signal951 (except clock signal), and processed signal 952 using phase lock-intechnique. In this arrangement, a probe signal generator 944 is used toperturb the biological subject 911 (e.g., heating 911 up using anoptical beam, or adding an electrical charge to 911), and response tothe probe signal is subsequently measured as a function of time using anarray of detection probes 955. The filtered signal in 952 shows dynamicresponse to probe signal by 944 as it decays over time. Since normalcell and abnormal cell may respond differently to the probe signal, thisarrangement with proper micro-probes can be utilized to detect diseasessuch as cancer. In another embodiment utilizing this set-up (shown inFIG. 14), the probe signal generator 944 can send a periodic signal tothe biological subject 911, detected response signal from the biologicalsubject by the detection probe 955 can be processed using the phaselock-in technique, with noise un-synchronized to the frequency of theprobe signal filtered out and signal synchronized to the probe signalfrequency amplified.

FIG. 15 is a perspective illustration of the novel multi-propertymicro-filter. A timed shutter 1502 is sandwiched between 2 pieces offilter membrane 1501 with wells. When a biological subject 1511 movesthrough the pathway of the well, it is first detected by the counter1512, which triggers the clock of the barrier panel 1502. Then thelarger cells will be filtered out, or blocked, by the filter's holes1001, while only the specific subjects with enough speed are able to getthrough the pathway 1503 before the timed shutter 1502 closes the filterpathway (see FIG. 15(b)). Otherwise it will be held back as the timedshutter 1502 moves to block the pathway as shown in FIG. 15(c).

FIG. 16 illustrates a fluid delivery system that includes a pressuregenerator, a pressure regulator, a throttle valve, a pressure gauge, anddistributing kits. The pressure generator 1605 sustains fluid withdesired pressure, and the pressure is further regulated by the regulator1601 and then accurately manipulated by the throttle valve 1602.Meanwhile, the pressure is monitored at real time and fed back to thethrottle valve 1602 by the pressure gauge 1603. The regulated fluid isthen in parallel conducted into the multiple devices where a constantpressure is needed to drive the fluid sample.

FIG. 17 illustrates how a micro-device in a disease detection apparatusof this invention can communicate, probe, detect, and optionally treatand modify biological subjects at a microscopic level. FIG. 17(a)illustrates the sequence of cellular events from signal recognition tocell fates determination. First, as the signals 1701 are detected byreceptors 1702 on the cell surface, the cell will integrate and encodethe signals into a biologically comprehensible message, such as calciumoscillation 1703. Consequently, corresponding proteins 1704 in the cellwill interact with the message, then be modified and transform intoion-interacted proteins 1705 accordingly. Through the translocation,these modified proteins 1705 will pass the carried message to thenuclear proteins, and the controlled modification on nuclear proteinswill modulate the expression of gene 1707 which includes transcription,translation, epigenetic processes, and chromatin modifications. Throughmessenger RNA 1709, the message is in turn passed to specific proteins1710, thereby changing their concentration—which then determines orregulates a cell's decision or activities, such as differentiation,division, or even death.

FIG. 17(b) illustrates an apparatus of this invention which is capableof detecting, communicating with, treating, modifying, or probing asingle cell, by a contact or non-contact means. The apparatus isequipped with micro-probes and micro-injectors which are addressed andmodulated by the controlling circuitry 1720. Each individualmicro-injector is supplied with a separate micro-cartridge, whichcarries designed chemicals or compounds.

To illustrate how an apparatus of this invention can be used to simulatean intracellular signal, calcium oscillation is taken as an examplemechanism. First, a Ca²⁺-release-activated channel (CRAC) has to beopened to its maximal extent, which could be achieved by variousapproaches. In an example of the applicable approaches, a biochemicalmaterial (e.g., thapsigargin) stored in the cartridge 1724 is releasedby an injector 1725 to the cell, and the CRAC will open at the stimulusof the biological subject. In another example of the applicableapproaches, the injector 1724 forces a specific voltage on cellmembrane, which causes the CRAC to open as well.

The Ca²⁺ concentration of a solution in the injector 1728 can beregulated as it is a desirable combination of a Ca²⁺-containing solution1726, and a Ca²⁺ free solution 1727. While the injector 1730 contains aCa²⁺ free solution, then injectors 1728 and 1730 are alternatelyswitched on and off at a desired frequency. As such, the Ca²⁺oscillation is achieved and the content inside the cell membrane arethen exposed to a Ca²⁺ oscillation. Consequently, the cell's activitiesor fate is being manipulated by the regulated signal generated by theapparatus.

Meanwhile, the cell's response (e.g., in the form of an electric,magnetic, electromagnetic, thermal, optical, acoustical, or mechanicalproperty) can be monitored and recorded by the probes integrated in thisapparatus.

FIG. 17(c) illustrates another design of apparatus which is able tosetup communication with a single cell. The apparatus is equipped withmicro-probes which are coated with biologically compatible compounds orelements, e.g., Ca, C, Cl, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na,S, or Zn. These probes can generate oscillating chemical signals withsuch an element or compound to interact with the cell, and results intoa response that affects the cell's activities or eventual fate asdescribe above. Likewise, this apparatus can probe and record the cell'sresponse (e.g., in the form of an electric, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical property) aswell.

FIG. 18 illustrates the system block diagram of a disease detectionapparatus of this invention. This example includes a fluid deliveringsystem 1801, biological interface 1802, a probing and detecting device1803, a system controller 1805, a medical waste reclaiming and treatingsystem 1804. A biological sample or material is transported to theinterface 1802 by the fluid delivery system 1801, meanwhile the fluidparameters (or properties) are reported to the system controller 1805which comprises a logic processing unit, a memory unit, an applicationspecific chip, a sensor, a signal transmitter, and a signal receiver;and then the system controller 1805 can give further command to thesystem. The interface 1802 is an assembly which bridges a fluid sampleand the detecting device, and further monitors the parameters orproperties of the biological sample (e.g., pressure, temperature,stickiness, or flow rate) and then reports the date to the systemcontroller 1805 while distributing the biological sample to the probingand detecting device 1803 with a specified speed or pressure (which canbe commanded by the system controller 1805).

The system controller 1805 is the central commander and monitor of theentire system (or apparatus), where all the parameters and informationfrom various modules is processed and exchanged and the instructions aregiven out, and where the command is dispatched. The system controller1805 can include, e.g., a pre-amplifier, an electrical meter, a thermalmeter, a switching matrix, a system bus, a nonvolatile storage device, arandom access memory, a processor, and a user interface through whichthe user of the apparatus can manipulate, configure the apparatus, andread the operating parameters and final result. The pre-amplifier canprocess the raw signal to a recognizable signal for the meters. Themeters can force and measure corresponding signals which can be, e.g.,electric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical signals, orcombinations thereof. The switching matrix can switch the testingterminals of different arrays of the probe sub-apparatus. The userinterface includes input and output assemblies and is an assembly whichseals the fluid delivery system and the probing and detecting devicetogether.

The probing and detecting device 1803 is the core functional module ofthe disease detection apparatus of this invention as it is the unit thatprobes the biological sample and collects related cellular signals (orresponses). The waste reclaiming and treating system 1804 reclaims thewaste biological sample to protect the privacy of its biological host,and keeps it away from polluting the environment.

FIGS. 19(b)-(n) illustrate a process flow for fabricating a micro-devicefor trapping, sorting, probing, measuring, treating, or modifying abiological subject (e.g., a single cell, a DNA or RNA molecule). A firstmaterial 1902 (e.g., a piezo-electric conducting material) and a secondmaterial 1903 (e.g., a conducting material) are sequentially depositedon a substrate 1901 (see FIGS. 19(b) and 19(c)). The second material1903 is subsequently patterned by lithography and etch processes (seeFIG. 19(d)). A third material 1904 is next deposited (as shown in FIG.19(e)) and planarized (see FIG. 19(f)). A layer of a fourth material1905 is subsequently deposited (see FIG. 19(g)) and patterned as a hardmask (see FIG. 19(h)), followed by etch to remove the third and firstmaterials from desired areas, which stops on the substrate 1901. FIG.19(i) is a perspective illustration of the device, while FIG. 19(j) is avertical illustration of the same device.

FIG. 19 (k) illustrates the use of a micro-device capable of trapping aDNA 1920 and measuring various properties (e.g., electrical, magnetic,physical, thermal, chemical, biological, bio-chemical, or opticalproperties) of a DNA. Each probe tip 1912 matches up spatially witheither a major groove or minor groove of a double helix DNA. Meanwhile,two probes (1911 and 1910) configured at the end of the trench can forceor measure signals to each strand end of the DNA's double helix. Theprobes can be made of a conducting material with optionally apiezo-electric support structure, which can stretch forward and backwardat a desired distance. All the probes are numbered, addressed, andcontrolled by a controlling circuitry.

FIG. 19(l) shows a simplified form of the device illustrated in FIG.19(k). In this device, probe tips match spatially with interlacedgrooves of a double helix DNA. The number of groove intervals betweenthe adjacent probes is variable. If required, either DNA can be moved(for example, by pulling by probes 1910 and 1911) or the probes can movealong the trench direction, mapping out properties in a full or partialDNA.

FIG. 20 illustrates an apparatus of this invention that is capable ofdetecting or measuring the surface charge of a biological subject 2010.It includes a channel, a pair of plates 2022, and a slit 2030 whichseparates the channel into a top channel 2041 and a bottom channel 2051.When a biological subject 2010 carrying a surface charge (positivecharge shown in FIG. 20(a)) passes through the channel, under theinfluence of the voltage applied on the plates 2022 (with positivevoltage at the top plate and negative at the bottom plate), it will movetowards the bottom plate as shown in FIG. 20(b). Thus, the biologicalsubject 2010 will pass through the bottom channel 2051 when it reachesslit 2030. (If the biological subject 2010 carries a negative charge, itwould pass through the top channel 2041.) This way, a biological subjectwith unknown charge type (negative or positive) can be determined byusing this apparatus.

This device comprises at least 2 parts of channel, one of which ischannel 2060 where the biological subject is charged or modified, andthe other comprises at least one plate or slit to separate thebiological subjects (e.g., where the biological subjects are separated).

As surface charge will affect the shape of a biological subject, byusing novel and multiple plates, information on the shape and chargedistribution of biological subjects can be obtained. The generalprinciple and design of the micro-device can be extended to a broaderscope, thereby making it possible to obtain other information on thebiological subject via separation by applying other parameters such asion gradient, thermal gradient, optical beam, or another form of energy.

FIG. 21 illustrates another apparatus of this invention for detecting ormeasuring microscopic properties of a biological subject 2110 byutilizing a micro-device that includes a channel, a set of probes 2120,and a set of optical sensors 2132 (see, FIG. 21(a)). The detectedsignals by probes 2120 can be correlated to information including imagescollected by the optical sensors 2132 to enhance detection sensitivityand specificity. The optical sensors can be, e.g., a CCD camera, aflorescence light detector, a CMOS imaging sensor, or any combination.

Alternatively, a probe 2120 can be designed to trigger optical emissionsuch as florescence light emission 2143 in the targeted biologicalsubject such as diseased cells, which can then be detected by an opticalprobe 2132 as illustrated in FIG. 21(c). Specifically, biologicalsubjects can be first treated with a tag solution which can selectivelyreact to diseased cells. Subsequently, upon reacting (contact ornon-contact) with probe 2120, optical emissions from diseased cellsoccur and can be detected by optical sensors 2132. This novel processusing the micro-devices of this invention is more sensitive than suchconventional methods as traditional florescence spectroscopy as theemission trigger point is directly next to the optical probe and thetriggered signal 2143 can be recorded in real time and on-site, withminimum loss of signal.

FIG. 22 illustrates another embodiment of the apparatus of thisinvention, which can be used to separate biological subjects ofdifferent geometric size and detect their properties respectively. Itincludes at least an entrance channel 2210, a disturbing fluid channel2220, an accelerating chamber 2230, and two selecting channels 2240 and2250. The angle between 2220 and 2210 is between 0° and 180°. Thebiological subject 2201 flows in the x-direction from 2210 to 2230. Thebiocompatible distribution fluid 2202 flows from 2220 to 2230. Then thefluid 2202 will accelerate 2201 in y-direction. However, theacceleration correlates with the radius of the biological subjects andthe larger ones are less accelerated than the small ones. Thus, thelarger and smaller subjects are separated into different channels.Meanwhile, probes can be optionally assembled aside the sidewall of2210, 2220, 2230, 2240, and 2250. They could detect electric, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,physical, or mechanical properties at the microscopic level. In the meantime, if desired, a cleaning fluid can also be injected into the systemfor dissolving and/or cleaning biological residues and deposits (e.g.,dried blood and protein) in the narrow and small spaces in theapparatus, and ensuring smooth passage of a biological subject to betested through the apparatus.

The channel included in the apparatus of this invention can have a widthof, e.g., from 1 nm to 1 mm. The apparatus should have at least oneinlet channel and at least two outlet channels.

FIG. 23 shows another apparatus of this invention with an acousticdetector 2320 for measuring the acoustic property of a biologicalsubject 2301. This apparatus includes a channel 2310, and at least anultrasonic emitter and an ultrasonic receiver installed along thesidewall of the channel. When the biological subject 2301 passes throughthe channel 2310, the ultrasonic signal emitted from 2320 will bereceived after carrying information on 2301 by the receiver 2330. Thefrequency of the ultrasonic signal can be, e.g., from 2 MHz to 10 GHz,and the trench width of the channel can be, e.g., from 1 nm to 1 mm. Theacoustic transducer (i.e., the ultrasonic emitter) can be fabricatedusing a piezo-electric material (e.g., quartz, berlinite, gallium,orthophosphate, GaPO₄, tourmalines, ceramics, barium, titanate, BatiO₃,lead zirconate, titanate PZT, zinc oxide, aluminum nitride, andpolyvinylidene fluorides).

FIG. 24 shows another apparatus of this invention that includes apressure detector for biological subject 2401. It includes at least onechannel 2410 and whereon at least one piezo-electric detector 2420. Whenthe biologic subject 2401 passes through the channel, the piezo-electricdetector 2420 will detect the pressure of 2401, transform theinformation into an electrical signal, and send it out to a signalreader. Likewise, the trench width in the apparatus can be, e.g., from 1nm to 1 mm, and the piezo-electric material can be, e.g., quartz,berlinite, gallium, orthophosphate, GaPO₄, tourmalines, ceramics,barium, titanate, BatiO₃, lead zirconate, titanate PZT, zinc oxide,aluminum nitride, or polyvinylidene fluorides.

FIG. 25 shows another apparatus of this invention that include a concavegroove 2530 between a probe couple, in the bottom or ceiling of thechannel. When a biological subject 2510 passes through, the concave 2530can selectively trap the biological subject with particular geometriccharacteristics and makes the probing more efficiently. The shape ofconcave's projection can be rectangle, polygon, ellipse, or circle. Theprobe could detect electric, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, physical, or mechanicalproperties. Similarly, the trench width can be, e.g., from 1 nm to 1 mm.FIG. 25(a) is an up-down view of this apparatus, FIG. 25(b) is a sideview, whereas FIG. 25(c) is a perspective view.

FIG. 26 is another apparatus of this invention that also includesconcave grooves 2630 (of a different shape from those shown in FIG. 25)on the bottom or ceiling of the channel. When a biological subject 2610passes through, the concave grooves 2630 will generate a turbulentfluidic flow, which can selectively trap the micro-biological subjectswith particular geometric characteristics. The probe could detectelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, physical, or mechanical properties. The depth ofthe concave groove can be, e.g., from 10 nm to 1 mm, and the channelwidth can be, e.g., from 1 nm to 1 mm.

FIG. 27 illustrated an apparatus of this invention with a steppedchannel 2710. When a biological subject 2701 passes through the channel2710, probe couples of different distances can be used to measuredifferent microscopic properties, or even the same microscopic atdifferent sensitivity at various steps (2720, 2730, 2740) with probeaside each step. This mechanism can be used in the phase lock-inapplication so that signal for the same microscopic property can beaccumulated. The probes can detect or measure microscopic electric,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, physical, or mechanical properties.

FIG. 28 illustrates another apparatus of this invention with thermalmeters 2830. It includes a channel, a set of probes 2820, and a set ofthermal meters 2830. The thermal meters 2830 can be an infrared sensor,a transistor sub-threshold leakage current tester, or thermister.

FIG. 29 illustrates a specific apparatus of this invention whichincludes carbon a nano-tube 2920 with a channel 2910 inside, probes 2940which can detect microscopic electric, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, physical, ormechanical properties. The carbon nano-tube 2920 as shown contains adouble-helix DNA molecule 2930. The carbon nano-tube can force and senseelectrical signals by the probes 2940 aside. The diameter of the carbonnano tube diameter can be, e.g., from 0.5 nm to 50 nm, and its lengthcan range from, e.g., 5 nm to 10 mm.

FIG. 30 shows an integrated apparatus of this invention that includes adetecting device (shown in FIG. 30(a)) and an optical sensor (shown inFIG. 30(b)) which can be, e.g., a CMOS image sensor (CIS), aCharge-Coupled Device (CCD), a florescence light detector, or anotherimage sensor. The detecting device comprises at least a probe and achannel, and the image device comprises at least 1 pixel. FIG. 30(c-1)and FIG. 30(c-2) illustrate the device with the detecting device andoptical sensor integrated. As illustrated in FIG. 30(d), when biologicalsubjects 3001, 3002, 3003 pass through, the probe 3010 in the channel3020, its electric, magnetic, electromagnetic, thermal, optical,acoustical, biological, chemical, physical, or mechanical property couldbe detected by the probe 3010 (see FIG. 30(e)), meanwhile its imagecould be synchronously recorded by the optical sensor (FIG. 30(f)). Boththe probed signal and image are combined together to provide a diagnosisand enhanced detection sensitivity and specificity. Such a detectingdevice and an optical sensing device can be designed in a system-on-chipor be packaged into one chip.

FIG. 31 shows an apparatus with a detecting micro-device (FIG. 31(a))and a logic circuitry (FIG. 31(b)). The detecting device comprises atleast a probe and a channel, and the logic circuitry comprises anaddressor, an amplifier, and a RAM. When a biological subject 3101passes through the channel, its property could be detected by the probe3130, and the signal can be addressed, analyzed, stored, processed, andplotted in real time. FIG. 31(c-1) and FIG. 31(c-2) illustrate thedevice with detecting device and Circuitry integrated. Similarly, thedetecting device and the integrated circuit can be designed in aSystem-on-Chip or be packaged into one chip.

FIG. 32 shows an apparatus of this invention that comprises a detectingdevice (FIG. 32(a)) and a filter (FIG. 32(b)). When a biological subject3201 passes through the device, a filtration is performed in the filter,and irrelevant objects can be removed. The remaining subjects' propertycan then be detected by the probe device (FIG. 31(a)). The filtrationbefore probing will enhance the precision of the device. The width ofthe channel can also range, e.g., from 1 nm to 1 mm.

FIG. 33 shows the geometric factors of DNA 3330 such as spacing in DNA'sminor groove (3310) have an impact on spatial distribution ofelectrostatic properties in the region, which in turn may impact localbiochemical or chemical reactions in the segment of this DNA. Byprobing, measuring, and modifying spatial properties of DNA (such as thespacing of minor groove) using the disclosed detector and probe 3320,one may detect properties such as defect of DNA, predictreaction/process at the segment of the DNA, and repair or manipulategeometric properties and therefore spatial distribution of electrostaticfield/charge, impacting biochemical or chemical reaction at the segmentof the DNA. For example, tip 3320 can be used to physically increasespacing of minor groove 3310.

FIG. 34 shows the fabrication process for a micro-device of thisinvention that has a flat cover atop of trench to form a channel. Thiswill eliminate the need for coupling two trenches to form a channel,which can be tedious for requiring perfect alignment. The cover can betransparent and allow observation with a microscope. It can comprise orbe made of silicon, SiGe, SiO₂, various types of glass, or Al₂O₃.

FIG. 35 is a diagram of an apparatus of this invention for detecting adisease in a biological subject. This apparatus includes apre-processing unit, a probing and detecting unit, a signal processing,and a disposal processing unit.

FIG. 36 shows an example of a sample filtration sub-unit in thepre-processing unit, which can separate the cells with differentdimensions or sizes. This device comprises at least one entrance channel3610, one disturbing fluid channel 3620, one accelerating chamber 3630,and two selecting channels (3640 and 3650). The angle 3660 between 3620and 3610 ranges from 0° to 180°.

The biological subject 3601 flows in the x direction from the entrancechannel 3610 to the accelerating chamber 3630. A bio-compatible fluid3602 flows from disturbing fluid channel 3620 to the acceleratingchamber 3630, it then accelerates the biological subject 3601 in they-direction. The acceleration correlates with the radius of thebiological subject and the larger ones are less accelerated than thesmaller ones. Then, the larger and smaller subjects are separated intodifferent selecting channels. Meanwhile, probes can be optionallyassembled on the sidewalls of the channels 3610, 3620, 3630, 3640, and3650. The probes could detect, at the microscopic level, electric,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, biochemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, physical, or mechanical properties.

FIG. 37 is a diagram of another example of a sample filtration unit inthe apparatus of this invention. 3701 represents small cells, while 3702represents large cells. When a valve 3704 is open and another valve 3703is closed, biological subjects (3701 and 3702) flow towards exit A.Large cells that have larger size than the filtration hole are blockedagainst exit A, while small cells are flushed out through exit A. Theentrance valve 3704 and exit A valve 3707 are then closed, and abio-compatible fluid is injected through the fluid entrance valve 3706.The fluid carries big cells are flushed out from exit B. The largercells are then analyzed and detected in the detection part of theinvention.

FIG. 38 is a diagram of a pre-processing unit of an apparatus of thisinvention. This unit includes a sample filtration unit, a rechargingunit or system for recharging nutrient or gas into the biologicalsubject, a constant pressure delivery unit, and a sample pre-probingdisturbing unit.

FIG. 39 is a diagram of an information or signal processing unit of anapparatus of this invention. This unit includes an amplifier (such as alock-in amplifier) for amplifying the signal, an A/D converter, and amicro-computer (e.g., a device containing a computer chip or informationprocessing sub-device), a manipulator, a display, and networkconnections.

FIG. 40 shows the integration of multiple signals which results incancellation of noise and enhancement of signal/noise ratio. In thisfigure, a biological 4001 is tested by Probe 1 during Δt between t1 andt2, and by Probe 2 during At between t3 and t4. 4002 is 4001′s testedsignal from Probe 1, and 4003 is from Probe 2. Signal 4004 is theintegration result from signal 4002 and 4003. The noise cancels out eachother in certain extent and results in an improved signal strength orsignal/noise ratio. The same principle can be applied to data collectedfrom more than more than 2 micro-devices or probing units.

FIG. 41 shows one embodiment of the fabrication processes flow of thisinvention for manufacturing a detection device with at least onedetection chamber and at least one detector. In this example, followingan optional process flow of fabricating data storage, data processingand analyzing components (including transistors, memory devices, logiccircuits, and RF devices), a material 4122 is first deposited onto asubstrate 4111, followed by the deposition of another material 4133(material for future detectors). Material 4133 can be selected fromelectrically conductive materials, piezo-electric materials,semiconductor materials, thermal sensitive materials, ion emissionsensitive materials, pressure sensitive materials, mechanical stresssensitive materials, or optical materials. Optionally, it can alsoconsist of composite materials or a desired material stack. If required,an integrated detector with a set of sub-components can be placed atthis level. Material 4133 is next patterned using lithography and etchprocesses, forming a set of desired features shown in FIG. 41(c).Another material 4144 is subsequently deposited, which can be the sameas or different from material 4122. Material 4122 can be an electricallyinsulating material such as oxide (SiO₂), doped oxide, silicon nitride,or polymer material. Next, the material 4144 is optionally planarizedusing polishing (e.g., using chemical mechanical polishing) or etch backprocess. The material stack is then patterned using lithography and etchprocesses, stopping on substrate 4111. Finally, as shown in FIG. 41(g),a capping layer or the surface of another component 4155 is placed ontop of the material stack (thereby sealing or capping it), forming anenclosed detection chamber 4166 with detector 4177 for biological sampledetection.

FIG. 42 illustrates another embodiment of the fabricating method of thisinvention for manufacturing a detection device with enclosed detectionchambers, detectors, and channels for transporting biological samplessuch as fluidic samples. In this embodiment, following an optionalprocess flow of fabricating data storage, data processing and analyzingcomponents (including transistors, memory devices, logic circuits, andRF devices), a material 4222 is first deposited onto a substrate 4211,followed by the deposition of another material 4233 (material for futuredetectors). Material 4233 can be selected from electrical conductivematerials, piezo-electric materials, semiconductor materials, thermalsensitive materials, ion emission sensitive materials, pressuresensitive materials, mechanical stress sensitive materials, or opticalmaterials. Optionally, it can also include composite materials or adesired material stack. If required, an integrated detector with a setof sub-components can be placed at this level.

Materials 4222 and 4233 are subsequently patterned using lithography andetch processes (FIG. 42(c)). These two layers (4222 and 4233) can bepatterned in separate patterning processes sequentially, or can bepatterned in the same process, pending on device design, types ofmaterials and etch chemistries. Substrate 4211 is next etched as shownin FIG. 42(d), forming a recessed area (cavity) in 4211, in which stacks4222 and 4233 can be used as a hard mask during the etch process.

A material 4244 is deposited into the recessed area, and the portion ofthe material 4244 above the material 4233 is removed using a polishing(chemical or mechanical) or etch back process. Material 4244 can beselected from oxide, doped oxide, silicon nitride, and polymermaterials. A layer 4255 is then deposited onto material 4244 andpatterned to form small holes at selected locations. A wet or vapor etchis utilized next to remove material 4244, forming an enclosed detectionchamber 4266.

Optionally, as shown in FIG. 42(i), the material 4222 is also removedusing wet or vapor etch process, forming channels 4288 connectingvarious detection chambers, thus forming detection chambers with adetector 4277 lined with the walls of the detection chamber and withgaseous or fluidic biological samples flowing through the chambers.Finally, the top surface of the detection chamber is sealed with anotherlayer of material (e.g., 4255).

FIG. 43 shows a novel disease detection method of this invention inwhich at least one probe object is launched at a desired speed anddirection toward a biological subject, resulting in a collision. Theresponse(s) by the biological subject during and/or after the collisionis detected and recorded, which can provide detailed and microscopicinformation on the biological subject such as weight, density,elasticity, rigidity, structure, bonding (between different componentsin the biological subject), electrical properties such as electricalcharge, magnetic properties, structural information, and surfaceproperties. For example, for a same type of cell, it is expected that acancerous cell will experience a smaller traveling distance after thecollision than that of a normal cell due to its denser, greater weight,and possibly larger volume. As shown in FIG. 43(a), a probe object 4311is launched towards a biological subject 4322. After the collision withthe probe object 4311, the biological subject 4322 may be pushed(scattered) out a distance depending on its properties as shown FIG.43(b).

FIG. 43(c) shows a schematic of a novel disease detection device with aprobe object launch chamber 4344, an array of detectors 4333, a probeobject 4322 and a biological subject to be tested 4311. In general, atest object can be an inorganic particle, an organic particle, acomposite particle, or a biological subject itself. The launch chambercomprises a piston to launch the object, a control system interfaced toan electronic circuit or a computer for instructions, and a channel todirect the object.

FIG. 44 illustrates a novel fabrication process for forming multiplecomponents with different materials at the same device level. First, afirst material 4422 is deposited onto a substrate 4411 (see FIG. 44(a)),followed by the deposition of a second material 4433. The secondmaterial 4433 is next patterned to form at least a portion of recessedarea in the layer 4433, using lithography and etch processes (see FIG.44(c)). A third material 4444 is subsequently deposited. The thirdmaterial can be the same as or different from the second material 4422.

The third material directly above the second material is removed viaetch back and/or polishing (such as chemical mechanical polishing)processes (see FIG. 44(e)). Optionally, the third material is nextpatterned to form at least a portion of recessed area in layer 4444(FIG. 44(f)). A fourth material 4455 is then deposited. Optionally, theportion of the fourth material 4455 directly above the third material4444 or above both the second and third materials is removed via etchback and/or polishing (such as chemical mechanical polishing). The aboveprocess can keep repeating to form multiple features with the same ordifferent materials at the same device level. Hence, this process flowforms at least two components 4466 and 4477 with different materials orthe same materials at the same device level. For example, in oneembodiment, one component can be used as a prober and the other can beused as a detector.

FIG. 45 illustrates a method for detecting a disease in a biologicalsubject. A biological subject 4501 passes through the channel 4531 at aspeed v, and probe 4511 is a probe which can grossly detect theproperties of the biological subject at high speed.

Probe 4512 is a fine probing device which is coated by a piezo-electricmaterial. There is a distance ΔL between probe 4511 and probe 4512.

When the biological subjects are tested when getting through 4511, ifthe entity is identified to be a suspected abnormal one, the systemwould trigger the piezo-electric probe 4512 to stretch into the channeland probe particular properties after a time delay of Δt. And probe 4512retracts after the suspected entity passed through.

The probing device is capable of measuring at the microscopic level anelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject.

The width of the micro-channel can range from about 1 nm to about 1 mm.

FIG. 46 shows a process of detecting a disease in a biological subject.A biological subject 4601 passes through the channel 4631 at a speed v.Probe 4611 is a probe which can grossly detect the properties of thebiological subject at high speed. 4621 and 4622 are piezo-electricvalves to control the micro-channel 4631 and 4632. 4612 is a fineprobing device which can probe biological properties more particularly.4631 is flush channel to rush out normal biological subjects. 4632 isdetection channel where the suspected entities are fine detected in thischannel.

When a biological subject is tested while getting through 4611, if it isnormal, the valve 4621 of the flush channel is open, while the detectionchannel valve 4622 is closed, the biological subject is flushed outwithout a time-consuming fine detection.

When the biological subject is tested while getting through 4611, if itis suspected to be abnormal or diseased, the valve 4621 of the flushchannel is closed, while the detection channel valve 4622 is open, thebiological subject is conducted to the detection channel for a moreparticular probing.

The width of the micro-channel can range from about 1 nm to about 1 mm.

The probing device is capable of measuring at the microscopic level anelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject.

FIG. 47 illustrates an arrayed biological detecting device. As shown inFIG. 47(a), 4701 are arrayed micro-channels which can get through thefluidics and biological subjects. 4702 are probing devices embeddedaside the channels. The sensors are wired by bit-lines 4721 andword-lines 4722. The signals are applied and collected by the decoderR\row-select 4742 and decoder column select 4741. As illustrated in FIG.47(b), the micro-channel arrayed biological detecting device 4700 can beembedded in a macro-channel 4701. The micro-channel's dimension rangesfrom about 1 um to about 1 mm. The shape of the micro-channel can berectangle, ellipse, circle, or polygon.

The probing device is capable of measuring at the microscopic level anelectric, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject.

FIG. 48 illustrates a device of the current invention for diseasedetection. 4801 is inlet of the detecting device, and 4802 is the outletof the device. 4820 is the channel where the biological subjects passthrough. 4811 is the optical component of the detecting device.

As illustrated in FIG. 48(b), the optical component 4811 consists of anoptical emitter 4812 and an optical receiver 4813. The optical emitteremits an optical pulse (e.g. laser beam pulse), when the biologicalsubject 4801 passing through the optical component, and the opticalsensor detects the diffraction of the optical pulse, then identify themorphology of the entity.

FIG. 49 shows a schedule for fabricating a piezo-electric micro-detectorof this invention. Particularly, in FIG. 49(a), a substrate 4901 isdeposited sequentially with a wet etching stop layer 4902 of material A,and with a sacrificial layer 4903 of material B. The sacrificial layer4903 is then patterned by the lithography and etching processes. Shownin FIG. 49(b), a layer 4904 of piezo-electric material C is thendeposited onto the surface of the sacrificial layer 4903, and thenplanarized. As shown in FIG. 49 (c), the layer 4904 is then patterned bylithography and etching processes. A second sacrificial layer 4905(which can be the same as or different from material B) and a second wetetching stop layer 4906 (which can be the same as or different frommaterial A) are subsequently deposited, as shown in FIG. 49(d) and FIG.49(e). A patterning process using lithography and etching is performedthrough layers 4906 and 4905, and etching is stopped on thepiezo-electric layer 4904. It is followed by depositing a conductivelayer 4907 of material D is deposited, and then patterning theconductive layer. See FIG. 49(g). A patterning process is then followedand the etching stopped on the substrate, thereby forming a trench. SeeFIG. 49(h). An isotropic wet etch selective to material B is thenfollowed, giving rise to a piezo-electric probe (a cantilever) 4908. SeeFIG. 49(i).

FIG. 50 shows an example of the micro-device of this invention packagedand ready for integration with a sample delivery system and datarecording device. As illustrated in FIG. 50(a), the device 5001 isfabricated by micro-electronics processes described herein and has atleast a micro-trench 5011, a probe 5022, and a bonding pad 5021. Thesurface of the device's top layer can include SixOyNz, Si, SixOy, SixNy,or a compound containing the elements of Si, O, and N. Component 5002 isa flat glass panel. In FIG. 50(b), the flat panel 5002 is shown to bebonded with micro-device 5001 on the side of micro-trench. The bondingcan be achieved by a chemical, thermal, physical, optical, acoustical,or electrical means, or any combination thereof. FIG. 50(c) shows aconductive wire being bonded with the bonding pad from the side of thepads. As illustrated in FIG. 50(d), the device 5001 is then packaged ina plastic cube with only conducting wires exposed. In FIG. 50(e), aconical channel 5020 is carved through packaging material and connectingthe internal channel of the device. As illustrated in FIG. 50(f), thelarger opening mouth of the conical channel makes it operational andconvenient to mount a sample delivery injector with the device, therebybetter enabling the delivery of sample from an injector with relativelylarge size of injector needle into device with relatively smallchannels.

FIG. 51 shows another example of the micro-device of this inventionpackaged and ready for integration with a sample delivery system anddata recording device. As shown in FIG. 51(a), a micro-device 5100 isfabricated by one or more micro-electronics processes as described inInternational Application No. PCT/U.S. 2011/042637, entitled “Apparatusfor Disease Detection.” The micro-device 5100 has at least amicro-trench 5104, a probe 5103, a connecting port 5102, and a bondingpad 5105. On the top of the micro-device 5100, the surface layercomprises SixOyNz, Si, SixOy, SixNy, or a compound consisting of Si, O,and N. The surface layer can be covered, and thus the micro-device 5100is mounted, with a flat glass panel 5101. See FIG. 51(b). The mountingcan be by a chemical, thermal, physical, optical, acoustical, orelectrical means. As shown in FIG. 51(c), the conductive wire is bondedwith bonding pad from the side of the pads. FIG. 51(d) illustrates thatthe micro-device 5100 can then be packaged in a cube with onlyconducting wires exposed. The packaging cube can comprise a packagingmaterial such as plastic, ceramic, metal, glass, or quartz. As shown inFIG. 51(e), a tunnel 5141 is then drilled into the cube until the tunnelreaches the connecting port 5102.

Further, as shown in FIG. 51(f), the tunnel 5141 is then being connectedto other pipes which can delivery a sample to be tested into themicro-device 5100, and flush out the sample after the sample is tested.

FIG. 52 shows yet another example of the micro-device of this inventionpackaged and ready for integration with a sample delivery system anddata recording device. As illustrated in FIG. 52(a), device 5200 is amicro-fluidic device which has at least one micro-channel 5201. 5203 isa pipe that conducts a fluidic sample. The micro-channel 5201 and theconducting pipe 5203 are aligned and submerged in a liquid, for example,water. FIG. 52(b) illustrates that, when the temperature of the liquidin which the micro-device and conducting pipe are submerged, isdecreased to its freezing point or lower, the liquid solidifies into asolid 5204. As illustrated in FIG. 52(c), while the temperature of theliquid is maintained below the freezing point, the combination(including the solid 5204, the conducting pipe 5203, and the device5200) is enclosed into a packaging material 5205 whose meltingtemperature is higher than that of the solid 5204, with only theconducting pipe exposed. FIG. 52(d) shows that, after the temperature isincreased above the melting point of the solid 5204, the solid material5204 melts and becomes a liquid and is then exhausted from theconducting pipe 5203. The space 5206 wherein the solid material 5204once filled is now available or empty, and the channel 5201 and theconducting pipe 5203 are now connected through and sealed in the space5206.

While for the purposes of demonstration and illustration, the abovecited novel, detailed examples show how microelectronics and/ornano-fabrication techniques and associated process flows can be utilizedto fabricate highly sensitive, multi-functional, powerful, andminiaturized detection devices, the principle and general approaches ofemploying microelectronics and nano-fabrication technologies in thedesign and fabrication of high performance detection devices have beencontemplated and taught, which can and should be expanded to variouscombination of fabrication processes including but not limited to thinfilm deposition, patterning (lithography and etch), planarization(including chemical mechanical polishing), ion implantation, diffusion,cleaning, various materials, combination of processes and steps, andvarious process sequences and flows. For example, in alternativedetection device design and fabrication process flows, the number ofmaterials involved can be fewer than or exceed four materials (whichhave been utilized in the above example), and the number of processsteps can be fewer or more than those demonstrated process sequences,depending on specific needs and performance targets. For example, insome disease detection applications, a fifth material such as abiomaterial-based thin film can be used to coat a metal detection tip toenhance contact between the detection tip and a biological subject beingmeasured, thereby improving measurement sensitivity.

Applications for the detection apparatus and methods of this inventioninclude detection of diseases (e.g., in their early stage), particularlyfor serious diseases like cancer. Since cancer cell and normal celldiffer in a number of ways including differences in possible microscopicproperties such as electrical potential, surface charge, density,adhesion, and pH, novel micro-devices disclosed herein are capable ofdetecting these differences and therefore applicable for enhancedcapability to detect diseases (e.g., for cancer), particularly in theirearly stage. In addition micro-devices for measuring electricalpotential and electrical charge parameters, micro-devices capable ofcarrying out mechanical property measurements (e.g., density) can alsobe fabricated and used as disclosed herein. In mechanical propertymeasurement for early stage disease detection, the focus will be on themechanical properties that likely differentiate disease or cancerouscells from normal cell. As an example, one can differentiate cancerouscells from normal cells by using a detection apparatus of this inventionthat is integrated with micro-devices capable of carrying outmicro-indentation measurements.

Although specific embodiments of this invention have been illustratedherein, it will be appreciated by those skilled in the art that anymodifications and variations can be made without departing from thespirit of the invention. The examples and illustrations above are notintended to limit the scope of this invention. Any combination ofdetection apparatus, micro-devices, fabrication processes, andapplications of this invention, along with any obvious their extensionor analogs, are within the scope of this invention. Further, it isintended that this invention encompass any arrangement, which iscalculated to achieve that same purpose, and all such variations andmodifications as fall within the scope of the appended claims.

All publications or patent applications referred to above areincorporated herein by reference in their entireties. All the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings) may be replaced by alternative features servingthe same, equivalent or similar purpose, unless expressly statedotherwise. Thus, unless expressly stated otherwise, each featuredisclosed is one example of a generic series of equivalent or similarfeatures.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. All publications referenced herein are incorporated byreference in their entireties.

What is claimed is:
 1. A method for detecting a mechanical or electricproperty of a biological subject, comprising: providing a piezo-electricmicro-device comprising a substrate, a piezo-electric material forming aprobe with a supporting point in the micro-device, an electronicallyconductive material, a material that is neither piezo-electric norelectronically conductive, wherein the piezo-electric material is placedbetween the electronically conductive material and the material that isneither piezo-electric nor electronically conductive, and at least partof the material that is neither piezo-electric nor electronicallyconductive is placed between the substrate and the piezo-electricmaterial; wherein an end portion of the probe extends out from thesupporting point and the end portion is substantially free andsurrounded without contact, by the material that is neitherpiezo-electric nor electronically conductive; and wherein themicro-device further comprises a detector, and contacting thepiezo-electric micro-device with the biological subject to be detected,wherein the piezo-electric micro-device detects the mechanical,acoustical, or electric property of the biological subject uponcontacting it and converts the mechanical, acoustical, or electricproperty to generate an electric or mechanical response, andtransferring the electric, acoustical, or mechanical response thusgenerated through the electrically conductive material to a recordingdevice or data analyzer, wherein the detector is an acoustic signalreceiver and capable of detecting an acoustic signal.
 2. A method fordetecting a mechanical or electric property of a biological subject,comprising: providing a piezo-electric micro-device comprising asubstrate, a piezo-electric material forming a probe with a supportingpoint in the micro-device, an electronically conductive material, amaterial that is neither piezo-electric nor electronically conductive,wherein the piezo-electric material is placed between the electronicallyconductive material and the material that is neither piezo-electric norelectronically conductive, and at least part of the material that isneither piezo-electric nor electronically conductive is placed betweenthe substrate and the piezo-electric material; wherein an end portion ofthe probe extends out from the supporting point and the end portion issubstantially free and surrounded without contact, by the material thatis neither piezo-electric nor electronically conductive; and wherein themicro-device further comprises a detector, and contacting thepiezo-electric micro-device with the biological subject to be detected,wherein the piezo-electric micro-device detects the mechanical,acoustical, or electric property of the biological subject uponcontacting it and converts the mechanical, acoustical, or electricproperty to generate an electric or mechanical response, andtransferring the electric, acoustical, or mechanical response thusgenerated through the electrically conductive material to a recordingdevice or data analyzer, wherein the micro-device further comprises anadditional probe that can be piezo-electric or non piezo-electric, andthe method further comprising the following steps: applying an inputsignal to the probe to generate an acoustic signal; scanning thefrequency range of the acoustic signal; contacting the probe with thebiological subject, thereby launching the acoustic signal to thebiological subject and causing the biological subject to generate aresponse signal which is then detected by the detector; recording theresponse signal detected by the detector as a function of the frequencyof the acoustic signal launched by the probe; optionally amplifying therecorded signal by the detector; optionally amplifying the recordedsignal by the detector using a lock-in amplifier; optionally filteringout noise in the recorded signal by the detector unsynchronized to theinput acoustic signal by the probe; optionally analyzing the recordedsignal; and reaching a diagnosis conclusion.
 3. The method of claim 2,wherein the input signal is a pulsed or modulated acoustic signal; orthe response signal is recorded for its resonant frequency or acousticintensity, or frequency versus intensity spectrum of the biologicalsystem and its immediate surrounding.